Recombinant elastase proteins and methods of manufacturing and use thereof

ABSTRACT

The present invention relates to methods for the manufacture, purification, formulation, and use of biologically active recombinant elastase proteins. Described are recombinant methods for producing therapeutically useful elastase proteins, as are pharmaceutical compositions comprising said elastase proteins. Novel recombinant elastase proteins and protein preparations are also disclosed. Methods are described for treating and preventing diseases of biological conduits using pharmaceutical compositions containing the elastase proteins of the invention.

This application is a division of U.S. application Ser. No. 14/465,754,filed Aug. 21, 2014, now abandoned, which is a division of U.S.application Ser. No. 12/823,098, filed Jun. 24, 2010, which iscontinuation-in-part of U.S. application Ser. No. 12/746,509, U.S.national stage of international application no. PCT/US2008/085559, filedDec. 4, 2008, now U.S. Pat. No. 9,057,067, issued Jun. 16, 2015, whichis a continuation in part of U.S. application Ser. No. 12/327,809, filedDec. 3, 2008, now U.S. Pat. No. 8,501,449, issued Aug. 6, 2013, whichclaims the priority benefit of U.S. provisional application No.60/992,319, filed Dec. 4, 2007, the contents of each of which areincorporated by reference herein in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 18, 2016, isnamed PRT-004C-D1-D1-SL.txt and is 149,753 bytes in size.

1. FIELD OF THE INVENTION

The present invention relates to recombinant methods of manufacturingand formulating elastase proteins for use in treating and preventingdiseases of biological conduits. The present invention further relatesto novel recombinant elastase proteins and pharmaceutical compositionscontaining such proteins. Yet further, the present invention relates tothe use of pharmaceutical compositions comprising recombinant elastaseproteins for the treatment and prevention of diseases of biologicalconduits.

2. BACKGROUND OF THE INVENTION

Elastin is a protein capable of spontaneously recoiling after beingstretched. Cross-linked elastin is the major structural component ofelastic fibers, which confers tissue elasticity. A proteinase may benamed an elastase if it possesses the ability to solubilize mature,cross-linked elastin (Bieth, J G “Elastases: catalytic and biologicalproperties,” at pp. 217-320 (Mecham Edition, Regulation of MatrixAccumulation, New York, Academic Press, 1986). Several published patentapplications (WO 2001/21574; WO 2004/073504; and WO 2006/036804)indicate that elastase, alone and in combination with other agents, isbeneficial in the treatment of diseases of biological conduits,including biological conduits which are experiencing, or susceptible toexperiencing, obstruction and vasospasm. For elastase therapy of humansubjects, the use of a human elastase is desirable to reduce the risk ofimmune reaction to a non-human elastase.

To this date, however, there is no known commercially viable means ofproducing biologically active elastase in sufficiently pure form and insufficient quantities for clinical applications. Because elastases arepowerful proteases that can hydrolyze numerous proteins other thanelastin, the proteolytic activity of elastase poses potential obstaclesfor its recombinant production. For example, the activity of matureelastase can damage the host cell which is expressing it, degradeitself, or degrade agents used to assist in the production orcharacterization of the elastase.

Elastases are often expressed as preproproteins, containing a signalpeptide, an activation peptide, and a mature, active portion. Cleavageof the signal sequence upon secretion yields a proprotein that can havelittle or no enzymatic activity, and whose amino acid sequence containsthe amino acid sequence of an activation peptide and a mature protein.Generally, for recombinant expression, an inactive precursor may beexpressed instead of the mature active enzyme to circumvent damage tothe cell that expresses it. For example, U.S. Pat. No. 5,212,068describes the cloning of human pancreatic elastase cDNAs (referred totherein as elastase “IIA,” “IIIA” and “IIIB”). The various elastaseswere expressed as full-length cDNAs, including the native signalsequences, in mammalian COS-1 cells. In addition, engineered versions ofthe elastases, containing a B. subtilis signal sequence and aβ-galactosidase signal sequence, were also expressed in B. subtilis andE. coli, respectively. U.S. Pat. No. 5,212,068 also suggests expressingelastases in S. cerevisiae. Generally, working examples of elastaseexpression in U.S. Pat. No. 5,212,068 show low activity of the recoveredelastase or require an activation step involving treatment with trypsin,to generate the active elastase. In addition, the elastases were largelypresent in inclusion bodies when expressed in E. coli, and only smallportions of the expressed elastase were soluble and active. None of theelastase preparations described in U.S. Pat. No. 5,212,068 was purifiedto pharmaceutical grade.

Thus, there is a need in the art for recombinant manufacturing methodsthat allow the generation of therapeutic amounts of biologically activepharmaceutical grade elastases, and preferably avoid a trypsinactivation step that is costly for large-scale preparation and canresult in trypsin contamination of the final product. Administration ofan elastase containing trypsin to a patient could result in activationof the protease-activated receptors 1 and 2, which may reduce some ofthe beneficial effects of elastase treatment.

Citation or identification of any reference in Section 2 or in any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention provides novel, efficient methods of makingrecombinant elastase proteins and the use of the recombinant proteins incompositions, e.g., pharmaceutical compositions, elastase formulationsor unit dosages, for the treatment and prevention of diseases ofbiological conduits.

The present invention is directed to auto-activated proelastaseproteins, nucleic acids encoding auto-activated proelastase proteins,host cells comprising said nucleic acids, methods of makingauto-activated proelastase proteins and the use of auto-activatedproelastase proteins in the manufacture of mature, biologically activeelastase proteins, for example for use in pharmaceutical formulations.The term “auto-activated” (or “autoactivated”) is used hereininterchangeably with the terms “auto-activating,” “self-activating,” and“self-activated” and is not intended to imply that an activation stephas taken place. The term “activation” is used herein interchangeablywith the term “conversion” and is not intended to imply that a proteinresulting from “activation” necessarily possesses enzymatic activity.

As used hereinbelow, and unless indicated otherwise, the term “elastase”generally refers to mature elastase proteins with elastase activity aswell as immature elastase proteins, including immature proelastaseproteins (also referred to herein as elastase proproteins) and immaturepreproelastase proteins (also referred to herein as elastasepreproproteins).

Preferred elastases of the invention are type I pancreatic elastases,e.g., human type I pancreatic elastase and porcine type I pancreaticelastase. Type I pancreatic elastases are sometimes referred to hereinas “elastase-1,” “elastase I,” “elastase type 1,” “type 1 elastase” or“ELA-1.” Human type I pancreatic elastase is also referred to herein ashELA-1 or human ELA-1, and porcine type I pancreatic elastase is alsoreferred to herein as pELA-1 or porcine ELA-1.

A mature elastase protein of the invention typically has an amino acidsequence encoded by a naturally occurring elastase gene or a variant ofsuch sequence. Preferred sequence variants, including variantscontaining amino acid substitutions, are described herein. A proelastaseprotein is a largely inactive precursor of a mature elastase protein,and a preproelastase protein further contains a signal sequence forprotein secretion. Pre and pro sequences of the elastase proteins of theinvention are typically not native to the elastase genes encoding themature elastase proteins of the invention. Thus, in a sense, theimmature elastase proteins of the invention are “chimeric” proteins,with their mature portions encoded by a naturally-occurring elastasegene and their immature portions encoded by non-elastase gene sequences.

For ease of reference, the elastase proteins of the invention and theircore sequence components are depicted in FIG. 2. As shown in FIG. 2, theamino acid residues within the proelastase sequence that are N-terminalto the cleavage bond (i.e., the bond that is cleaved to yield matureelastase protein) are designated herein as PX, . . . P5, P4, P3, P2, andP1, where P1 is immediately N-terminal to the cleavage bond, whereasamino acids residues located to the C-terminus to the cleavage bond (andto the N-terminus) of the mature elastase protein are designated P1′,P2′, P3′, . . . PX′, where P1′ is immediately C-terminal to the cleavagebond and represents the N-terminal amino acid residue of the matureprotein. FIG. 2 also shows the following components:

(1) SIGNAL SEQUENCE: A sequence that increases the proportion ofexpressed molecules that are secreted from the cell. An exemplarysequence is amino acids 1-22 of SEQ ID NOS:50 or 51.

(2) PROPEPTIDE+SPACER: An optional, preferably a non-elastase,propeptide sequence (such as yeast α-factor propropeptide) that canfurther optionally include one or more spacer sequences (a yeastα-factor propeptide sequences and Kex2 and STE13 spacer sequences aredepicted in FIG. 1B). In a specific embodiment, the propeptide sequencedoes not include a spacer.

(3) ELASTASE PROPEPTIDE: Peptide that, when present on the N-terminalend of an elastase, renders the molecule inactive or less active ascompared to the corresponding mature elastase protein. The elastasepropeptide may be contiguous with the activation peptide or may containadditional amino acids relative to the activation peptide. Exemplaryelastase propeptide sequences are amino acids 1-10 of SEQ ID NOS:64 and69.

(4) ACTIVATION PEPTIDE: Used interchangeably herein with “activationsequence,” an activation peptide is a component of, and can becontiguous with, the elastase propeptide. As shown in FIG. 2, theactivation peptide contains amino acid residues P10 through P1. Anexemplary activation peptide consensus sequence is SEQ ID NO:80 or SEQID NO:121; other examples of activation peptide sequences are SEQ IDNOS: 23, 72 and 73.

(5) RECOGNITION SEQUENCE: A recognition sequence is a component of theelastase propeptide. As shown in FIG. 2, the recognition sequencecontains amino acid residues P3 through P1. Exemplary recognitionsequence consensus sequences are SEQ ID NOS:11-13, 93, and 119, and anexemplary recognition sequence is SEQ ID NO:20.

(6) CLEAVAGE DOMAIN: A region in the proelastase protein that spans thecleavage bond. As shown in FIG. 2, the cleavage domain contains aminoacid residues P5 through P3′. Exemplary cleavage domain consensussequences are SEQ ID NOS:74, 135 and 125. Exemplary cleavage domainsequences are SEQ ID NOS:42, 43, 48, 49, 53, 53, 54 and 55.

(7) CLEAVAGE SITE: Another region in the proelastase protein that alsospans the cleavage bond. As shown in FIG. 2, the cleavage site containsamino acid residues P4 through P4′. An exemplary cleavage site sequenceis SEQ ID NO:27.

(8) PREPROELASTASE PROTEIN: A protein that can comprise all of thecomponent parts. An exemplary preproelastase protein can comprise apeptides of SEQ ID NO:50, 51, 96, or 97 followed by an operably linkedprotein of SEQ ID NO:64 or SEQ ID NO:69.

(9) PROELASTASE PROTEIN: A protein that comprises mature elastaseprotein, an elastase propeptide, and the optional propeptide and spacersequences. Exemplary proelastase sequences are SEQ ID NOS:64 and 69.

(10) MATURE ELASTASE PROTEIN: A protein that when properly processeddisplays elastase activity. Exemplary mature sequences are SEQ ID NO:1(human) and SEQ ID NO:39 (porcine).

The elastase protein components can be considered modular buildingblocks of the elastase proteins, proelastase proteins and preproelastaseproteins. For example, the present invention provides a proelastaseprotein comprising the sequence of an elastase propeptide and a matureelastase protein. The elastase propeptide can contain an activationpeptide. The elastase propeptide can also contain an elastaserecognition sequence. The present invention also provides a proelastaseprotein comprising a cleavage domain or cleavage site in the regionspanning the junction between the elastase propeptide and the matureelastase protein. The proelastase proteins may further comprise a signalsequence for secretion. Such proteins are considered preproelastaseproteins. The preproelastase proteins may further comprise a yeast alphafactor propeptide and optionally a spacer sequence between the signalsequence and the elastase propeptide. The elastase proteins of theinvention may also contain components in addition to the core modularcomponents illustrated in FIG. 2. For example, an elastase protein cancontain an epitope tag or a histidine tag for purification. It shouldalso be noted that the elastase proteins of the invention need notcontain all the components depicted in FIG. 2, but generally contain atleast one of the components (including, by way of example but notlimitation, a mature elastase or a proelastase sequence) depicted inFIG. 2. Exemplary elastase proteins of the invention are set forth inembodiments 1-12, 28-39 and 68-69 in Section 8 below, includingexemplary type I proelastase proteins set forth in embodiments 13-27 inSection 8 below.

In certain aspects, the proelastase proteins of the disclosure have anelastase recognition sequence P3-P2-P1, wherein the amino acid residueat the P3 position is selected from:

-   -   (a) any of the 26 natural amino acid residues except proline or        glycine;    -   (b) alanine, leucine, isoleucine, methionine, lysine,        asparagine, histidine, threonine, or valine; and    -   (c) alanine, leucine, isoleucine, methionine, lysine,        asparagine, or valine;

wherein the amino acid residue at the P2 position independently selectedfrom:

-   -   (a) proline, alanine, leucine, isoleucine, glycine, valine,        histidine asparagine, or threonine;    -   (b) proline, alanine, leucine, isoleucine, glycine, valine,        asparagine, threonine;    -   (c) proline, alanine, leucine, isoleucine, glycine, valine,        threonine; and    -   (d) proline;

and wherein the amino acid residue at the P1 position is independentlyselected from:

-   -   (a) alanine, leucine, valine, isoleucine or serine;    -   (b) alanine, leucine, valine, isoleucine, asparagine or serine;        and    -   (c) alanine.

In other aspects, the proelastase proteins of the disclosure have apropeptide portion of an elastase cleavage domain P5-P4-P3-P2-P1,wherein the amino acid residue at the P5 position is selected from:

-   -   (a) any of the 26 natural amino acid residues;    -   (b) glutamate, histidine, proline, glycine, asparagine, lysine        or alanine,    -   (c) histidine or glutamate; and    -   (d) histidine,

wherein the amino acid at the P4 position is independently selectedfrom:

-   -   (a) any of the 26 natural amino acid residues except glycine,        lysine, phenylalanine, tyrosine, tryptophan, or arginine; and    -   (b) threonine, alanine, proline, or histidine,

wherein the amino acid residue at the P3 position is independentlyselected from:

-   -   (a) any of the 26 natural amino acid residues except proline or        glycine;    -   (b) alanine, leucine, isoleucine, methionine, lysine,        asparagine, histidine, threonine, or valine; and    -   (c) alanine, leucine, isoleucine, methionine, lysine,        asparagine, or valine;

wherein the amino acid residue at the P2 position is independentlyselected from:

-   -   (a) proline, alanine, leucine, isoleucine, glycine, valine,        histidine asparagine, or threonine;    -   (b) proline, alanine, leucine, isoleucine, glycine, valine,        asparagine, threonine;    -   (c) proline, alanine, leucine, isoleucine, glycine, valine,        threonine; and    -   (d) proline;

and wherein the amino acid residue at the P1 position is independentlyselected from:

-   -   (a) alanine, leucine, valine, isoleucine or serine;    -   (b) alanine, leucine, valine, isoleucine, asparagine or serine;        and    -   (c) alanine.

In certain aspects, the proelastase proteins of the disclosure have anelastase cleavage domain in which:

-   -   the amino acid residue at the P1′ position is alanine, leucine,        valine, isoleucine or serine, and is most preferably valine;    -   the amino acid residue at the P2′ position is glycine, alanine,        or valine, and is most preferably valine;    -   the amino acid residue at the P3′ position is glycine, valine,        threonine, phenylalanine, tyrosine or tryptophan, and is most        preferably glycine.

In other aspects, the proelastase proteins of the disclosure have anactivation peptide in which:

-   -   the amino acid residue at the P10 position is threonine;    -   the amino acid residue at the P9 position is glutamine or        histidine;    -   the amino acid residue at the P8 position is aspartate;    -   the amino acid residue at the P7 position is leucine; and    -   the amino acid residue at the P6 position is proline.

In certain preferred embodiments, a proelastase protein of thedisclosure has a histidine residue at the P5 position and/or a prolineresidue at the P2 position and/or an alanine residue at the P1 position.

The present invention provides proelastase proteins comprising (i)optionally, a signal sequence; (ii) an elastase activation peptidesequence comprising an elastase recognition sequence operably linked to(iii) an amino acid sequence of a type I mature elastase.

In certain aspects, the proelastase protein has an elastase recognitionsequence of SEQ ID NO:119 or SEQ ID NO:124.

In certain aspects, the proelastase protein has a cleavage domain thepropeptide portion of which comprises the amino acid sequence of SEQ IDNO:120.

In certain aspects, the proelastase protein has a cleavage domain of SEQID NO:123 or SEQ ID NO:125.

In certain aspects, the proelastase protein has an activation peptidesequence of SEQ ID NO:121.

In certain aspects, the proelastase proteins of the invention areisolated.

Nucleic acids encoding the elastase proteins of the invention, methodsfor producing and purifying the proteins, recombinant cells and cellculture supernatants, compositions comprising elastase proteins (e.g.,pharmaceutical compositions, unit dosages, formulations), the use of theproteins for therapeutic purposes and kits comprising the proteins,formulations, pharmaceutical compositions and unit doses are encompassedherein.

The present invention provides nucleic acids encoding proelastaseproteins, vectors comprising such nucleic acid, and host cellsgenetically engineered to express proelastase proteins, for example ahost cells into which a vector encoding a proelastase protein isintroduced.

The present invention provides a solution comprising a proelastaseprotein. The solution can be a buffer solution and/or contain cellculture components. In a specific embodiment, the solution is a buffersolution comprising phosphate and/or Tris base.

The present invention provides a cell culture supernatant comprising aproelastase protein.

The invention provides methods of producing a mature elastase protein,comprising subjecting a solution containing proelastase protein toconditions that produce mature elastase protein. The invention furtherprovides methods of producing a maturing elastase protein, comprising:(a) culturing a host cell engineered to express a proelastase proteinunder conditions in which the proelastase protein is produced; (b)recovering, and optionally purifying, the proelastase protein, and (c)subjecting a solution comprising said proelastase protein to conditionsthat produce mature elastase protein, thereby producing a matureelastase protein. In certain aspects, such conditions do not includecontacting the proelastase protein with trypsin and/or contacting saidproelastase protein with a catalytic amount of an elastase and/orsubjecting the solution to a pH of 6 to 11. In a specific embodiment,the solution comprises cell culture components, e.g., if it is a cellculture supernatant or is made from cell culture supernatant containingproelastase protein. In certain aspects, the methods further comprisethe step of lyophilizing the mature elastase protein and/or purifyingthe mature elastase protein.

Nucleic acids encoding the elastase proteins of the invention areexemplified in embodiments 40-67 in Section 8 below, including vectors(see, e.g., embodiments 70-72). Also exemplified in Section 8 arerecombinant cells (see, e.g., embodiments 73-84), cell supernatantscontaining elastase proteins (see, e.g., embodiment 88). Methods forproducing elastase proteins are exemplified in embodiments 89-224,261-276 and 347-373 in Section 8. Methods for producing elastaseformulations are exemplified in embodiments 225-260 in Section 8.Methods of producing pharmaceutical compositions are exemplified inembodiments 374-385 in Section 8. Pharmaceutical compositions comprisingelastase proteins are exemplified in embodiments 277-313 and 386 inSection 8, and unit dosages are exemplified in embodiments 413-420 inSection 8. Formulations of elastase proteins are exemplified inembodiments 324-346 in Section 8. The use of the elastase proteins fortherapeutic purposes is exemplified in embodiments 387-414 in Section 8.Kits comprising the proteins are exemplified in Section 8 by way ofembodiments 421-424.

Various aspects of the invention with respect to proelastase proteinswith SEQ ID NOS:64 and 69 are exemplified as Specific Embodiments inSection 8 below; however, such embodiments are applicable to otherelastase protein sequences disclosed herein.

The production methods described herein often include an activationstep, whereby the activation peptide is removed from the proelastasesequence/separated from the mature elastase sequence, thereby generatinga mature elastase protein. The activation steps described herein may beauto activation steps, i.e., carried out by an elastase activity, ornon-auto activation step, i.e., non-elastase mediated, e.g., carried outby trypsin.

In certain aspects, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence which encodes an elastaseprotein (including but not limited to a protein of any one of SEQ IDNOS: 6-9, 64-69, 88-91, or 98-103) comprising (i) an elastase propeptidecomprising an activation peptide sequence comprising an elastaserecognition sequence operably linked to (ii) the amino acid sequence ofa protein having elastase activity. The protein optionally furthercomprises a signal sequence, such as a yeast α-factor signal peptide andexemplified by the amino acid sequence of SEQ ID NO:34, operably linkedto said elastase propeptide. The α-factor is sometimes referred toherein as “alpha-factor” or “alpha mating factor” or “α-mating factor.”In certain specific embodiments, the protein comprises a non-elastasepropeptide such as yeast α-factor propeptide. In certain specificembodiments, the protein can comprise one or more spacer sequences.Spacer sequences can include, but are not limited to, Kex2 and STE13protease cleavage sites. In a specific embodiment, a Kex2 spacer can beused. In another embodiment, a Kex2 spacer can be operably linked toSTE13 spacers as shown in FIG. 1B. A signal peptide sequence and anon-elastase propeptide sequence is exemplified by the amino acidsequences of SEQ IDS NO:51 or 97. A peptide containing a signal peptidesequence, a non-elastase propeptide sequence, and a spacer sequence isexemplified by the amino acid sequences of SEQ IDS NO:50 or 96.

In other specific embodiments, the signal sequence is a mammaliansecretion signal sequence, such as a porcine or human type I elastase(used interchangeably with a type I pancreatic elastase) signalsequence.

Preferably, the elastase recognition sequence is a type I pancreaticelastase recognition sequence.

In specific embodiments, the protein having type I elastase activity isa mature human type I elastase, for example a protein of the amino acidsequence of SEQ ID NO:1, 4, 5, 84, or 87.

The present invention also provides a nucleic acid molecule comprising anucleotide sequence which encodes a protein comprising (i) a signalsequence operable in Pichia pastoris operably linked to (ii) anactivation sequence (including but not limited to an amino acid sequenceof SEQ ID NOS: 23, 72, 73, or 80) comprising a protease recognitionsequence (including but not limited to an amino acid sequence of any ofSEQ ID NOS:11-23 and 93 which in turn is operably linked to (iii) theamino acid sequence of a mature human type I elastase. In a preferredembodiment, the protease recognition sequence is a human type I elastaserecognition sequence, most preferably an elastase recognition sequenceof SEQ ID NO:20.

Proelastase proteins (optionally comprising a signal sequence and thusrepresenting preproelastase proteins) and mature elastase proteinsencoded by the nucleic acids of the invention are also provided, as arecompositions (e.g., pharmaceutical compositions, formulations and unitdosages) comprising said mature elastase proteins.

In a preferred embodiment, the proelastase protein or mature elastaseprotein does not have an N-terminal methionine residue. In anotherembodiment, however, the proelastase protein or mature elastase proteindoes have an N-terminal methionine residue.

Table 1 below summarizes the sequence identifiers used in the presentspecification. Preferred proteins of the invention comprise or consistof any of SEQ ID NOS:1-32, 34, 37-73, 77, 78, 82-92, and 98-105 listedin Table 1 below, or are encoded in part or entirely by the nucleotidesequences of SEQ ID NO:33 and SEQ ID NO:81.

TABLE 1 Summary of amino acid and nucleotide SEQ ID NOS. NUCLEOTIDE ORAMINO MOLECULE ACID SEQ ID NO Mature human elastase I, including first“valine,” with Amino Acid 1 possible polymorphism at position 220 (V orL) (numbering refers to position in context of preproprotein) Maturehuman elastase I, minus first “valine,” with possible Amino Acid 2polymorphism at position 220 (V or L) (numbering refers to position incontext of preproprotein) Mature human elastase I, minus first two“valines,” with Amino Acid 3 possible polymorphism at position 220 (V orL) (numbering refers to position in context of preproprotein) Maturehuman elastase I, with first “valine” substituted by Amino Acid 4“alanine,” with possible polymorphism at position 220 (V or L)(numbering refers to position in context of preproprotein) Mature humanelastase I (isotype 2), including first “valine” Amino Acid 5 Engineeredelastase proprotein no. 1 (pPROT42 variant), Amino Acid 6 with possiblepolymorphism at position 220 (V or L) (numbering refers to position incontext of preproprotein) Engineered elastase proprotein no. 2, withpossible Amino Acid 7 polymorphism at position 220 (V or L) (numberingrefers to position in context of preproprotein) Engineered elastaseproprotein no. 3, with possible Amino Acid 8 polymorphism at position220 (V or L) (numbering refers to position in context of preproprotein)Engineered elastase proprotein no. 4, with possible Amino Acid 9polymorphism at position 220 (V or L) (numbering refers to position incontext of preproprotein) Engineered elastase proprotein no. 5 (pPROT24trypsin Amino Acid 10 activated sequence), with possible polymorphism atposition 220 (V or L) (numbering refers to position in context ofpreproprotein) Consensus elastase recognition sequence 1 (PositionsAmino Acid 11 Xaa₁ = P3, Xaa₂ = P2, Xaa₃ = P1) Consensus elastaserecognition sequence 2 (Positions P3- Amino Acid 12 P2-P1) Consensuselastase recognition sequence 3 (Positions P3- Amino Acid 13 P2-P1)Elastase recognition sequence 1 (Positions P3-P2-P1) Amino Acid 14Elastase recognition sequence 2 (Positions P3-P2-P1) Amino Acid 15Elastase recognition sequence 3 (Positions P3-P2-P1) Amino Acid 16Wild-type trypsin recognition sequence (pPROT24) Amino Acid 17(Positions P3-P2-P1) Elastase recognition sequence 5 (PositionsP3-P2-P1) Amino Acid 18 Elastase recognition sequence 6 (PositionsP3-P2-P1) Amino Acid 19 Elastase recognition sequence 7 (PositionsP3-P2-P1 of Amino Acid 20 Variants 48 and 55) Elastase recognitionsequence 8 Amino Acid 21 Human elastase activation sequence 1 Amino Acid22 Human elastase activation sequence 2 Amino Acid 23 pro-PROT-201cleavage site Amino Acid 24 pPROT40 cleavage site Amino Acid 25 pPROT41cleavage site Amino Acid 26 pPROT42 cleavage site Amino Acid 27 pPROT43cleavage site Amino Acid 28 pPROT44 cleavage site Amino Acid 29 pPROT45cleavage site Amino Acid 30 pPROT46 cleavage site Amino Acid 31 pPROT47cleavage site Amino Acid 32 Coding region of a human elastase-1 (i.e.,human type I Nucleotide 33 pancreatic elastase) (NCBI Accession No.NM_001971) Yeast alpha factor signal peptide Amino Acid 34 20F primerNucleotide 35 24R primer Nucleotide 36 pPROT42 P3 Cleavage Site VariantElastase, with possible Amino Acid 37 polymorphism at position 220 (V orL) (numbering refers to position in context of preproprotein) pPROT42 P2Cleavage Site Variant Elastase, with possible Amino Acid 38 polymorphismat position 220 (V or L) (numbering refers to position in context ofpreproprotein) Mature porcine pancreatic Elastase I (from GenBank AminoAcid 39 Accession P00772.1) Elastase Variant Propeptide Cleavage Domain40 Amino Acid 40 Elastase Variant Propeptide Cleavage Domain 41 AminoAcid 41 Elastase Variant Propeptide Cleavage Domain 42 Amino Acid 42Elastase Variant Propeptide Cleavage Domain 43 Amino Acid 43 ElastaseVariant Propeptide Cleavage Domain 44 Amino Acid 44 Elastase VariantPropeptide Cleavage Domain 45 Amino Acid 45 Elastase Variant PropeptideCleavage Domain 46 Amino Acid 46 Elastase Variant Propeptide CleavageDomain 47 Amino Acid 47 Elastase Variant Propeptide Cleavage Domain 48Amino Acid 48 Elastase Variant Propeptide Cleavage Domain 49 Amino Acid49 Yeast alpha-mating factor signal peptide, propeptide, and Amino Acid50 spacer sequence 1 Yeast alpha-mating factor signal peptide andpropeptide Amino Acid 51 sequence 2 Elastase Variant Propeptide CleavageDomain 52 Amino Acid 52 Elastase Variant Propeptide Cleavage Domain 53Amino Acid 53 Elastase Variant Propeptide Cleavage Domain 54 Amino Acid54 Elastase Variant Propeptide Cleavage Domain 55 Amino Acid 55 ElastaseVariant Propeptide Cleavage Domain 56 Amino Acid 56 Elastase VariantPropeptide Cleavage Domain 57 Amino Acid 57 Elastase Variant PropeptideCleavage Domain 58 Amino Acid 58 Elastase Variant Propeptide CleavageDomain 59 Amino Acid 59 Elastase Variant Propeptide Cleavage Domain 60Amino Acid 60 Elastase Variant Propeptide Cleavage Domain 61 Amino Acid61 Elastase Variant Propeptide Cleavage Domain 62 Amino Acid 62 ElastaseVariant Propeptide Cleavage Domain 63 Amino Acid 63 Elastase Proenzymewith variant Cleavage Domain 48, with Amino Acid 64 possiblepolymorphism at position 220 (V or L) (numbering refers to position incontext of preproprotein) Elastase Proenzyme with variant CleavageDomain 49, with Amino Acid 65 possible polymorphism at position 220 (Vor L) (numbering refers to position in context of preproprotein)Elastase Proenzyme with variant Cleavage Domain 52, with Amino Acid 66possible polymorphism at position 220 (V or L) (numbering refers toposition in context of preproprotein) Elastase Proenzyme with variantCleavage Domain 53, with Amino Acid 67 possible polymorphism at position220 (V or L) (numbering refers to position in context of preproprotein)Elastase Proenzyme with variant Cleavage Domain 54, with Amino Acid 68possible polymorphism at position 220 (V or L) (numbering refers toposition in context of preproprotein) Elastase Proenzyme with variantCleavage Domain 55, with Amino Acid 69 possible polymorphism at position220 (V or L) (numbering refers to position in context of preproprotein)Wild Type Elastase + AlaArg Cleavage Variant, with Amino Acid 70possible polymorphism at position 220 (V or L) (numbering refers toposition in context of preproprotein) Wild Type Elastase + Arg CleavageVariant, with possible Amino Acid 71 polymorphism at position 220 (V orL) (numbering refers to position in context of preproprotein) Variant 48Human Elastase Activation peptide Amino Acid 72 Variant 55 HumanElastase Activation peptide Amino Acid 73 Human Elastase Cleavage DomainConsensus Sequence; Amino Acid 74 corresponds to residues P5, P4, P3,P2, P1, P′1, P′2, and P′3 of an elastase cleavage domain PCR MutagenesisPrimer for pPROT3 construction Nucleic Acid 75 PCR Mutagenesis Primer orpPROT3 construction Nucleic Acid 76 Mature ELA1 C-terminal Variant ofTalas et al, 2000, Amino Acid 77 Invest Dermatol. 114(1): 165-70. MatureELA-1 variants, with possible polymorphisms at Amino Acid 78 positions44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numberingrefers to position in context of preproprotein) Activation peptidevariants (wild type, trypsin cleavable), Amino Acid 79 with possiblepolymorphisms at position 10 (Q or H) (numbering refers to position incontext of preproprotein) Activation peptide “consensus” sequence AminoAcid 80 Coding region of ELA-1.2A Amino Acid 81 Translation Product ofELA-1.2A Amino Acid 82 (Trypsin activated pPROT24 sequence) TranslationProduct of ELA-1.2A Amino Acid 83 (trypsin activated pPROT24 sequence),with possible polymorphisms at positions 44 (W or R); 59 (M or V); 220(V or L); and 243 (Q or R) (numbering refers to position in context ofpreproprotein) Mature human elastase I, including first “valine,” withAmino Acid 84 possible polymorphisms at positions 44 (W or R); 59 (M orV); 220 (V or L); and 243 (Q or R) (numbering refers to position incontext of preproprotein) mature human elastase I, minus first “valine,”with possible Amino Acid 85 polymorphisms at positions 44 (W or R); 59(M or V); 220 (V or L); and 243 (Q or R) (numbering refers to positionin context of preproprotein) Mature human elastase I, minus first two“valines,” with Amino Acid 86 possible polymorphisms at positions 44 (Wor R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers toposition in context of preproprotein) mature human elastase I, withfirst “valine” substituted by Amino Acid 87 “alanine,” with possiblepolymorphisms at positions 44 (W or R); 59 (M or V); 220 (V or L); and243 (Q or R) (numbering refers to position in context of preproprotein)Engineered elastase proprotein no. 1 (pPROT42 variant), Amino Acid 88with possible polymorphisms at positions 10 (Q or H); 44 (W or R); 59 (Mor V); 220 (V or L); and 243 (Q or R) (numbering refers to position incontext of preproprotein) Engineered elastase proprotein no. 2, withpossible Amino Acid 89 polymorphisms at positions 10 (Q or H); 44 (W orR); 59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers toposition in context of preproprotein) Engineered elastase proprotein no.3, with possible Amino Acid 90 polymorphisms at positions 10 (Q or H);44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numberingrefers to position in context of preproprotein) engineered elastaseproprotein no. 4, with possible Amino Acid 91 polymorphisms at positions10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R)(numbering refers to position in context of preproprotein) engineeredelastase proprotein no. 5 (pPROT24 trypsin Amino Acid 92 activatedsequence), with possible polymorphisms at positions 10 (Q or H); 44 (Wor R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers toposition in context of preproprotein) Consensus elastase recognitionsequence 4 (Positions P3- Amino Acid 93 P2-P1) pPROT42 P3 Cleavage SiteVariant Elastase, with possible Amino Acid 94 polymorphisms at positions44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numberingrefers to position in context of preproprotein) pPROT42 P2 Cleavage SiteVariant Elastase, with possible Amino Acid 95 polymorphisms at positions44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R) (numberingrefers to position in context of preproprotein) Yeast alpha-matingfactor signal peptide, propeptide, and Amino Acid 96 spacer sequence 1Yeast alpha-mating factor signal peptide and propeptide Amino Acid 97sequence 2 Elastase Proenzyme with variant Cleavage Domain 48 Amino Acid98 Generic to SEQ ID NO: 64, with possible polymorphisms at positions 10(Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R)(numbering refers to position in context of preproprotein) ElastaseProenzyme with variant Cleavage Domain 49, with Amino Acid 99 possiblepolymorphisms at positions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (Vor L); and 243 (Q or R) (numbering refers to position in context ofpreproprotein) Elastase Proenzyme with variant Cleavage Domain 52, withAmino Acid 100 possible polymorphisms at positions 10 (Q or H); 44 (W orR); 59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers toposition in context of preproprotein) Elastase Proenzyme with variantCleavage Domain 53, with Amino Acid 101 possible polymorphisms atpositions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and 243(Q or R) (numbering refers to position in context of preproprotein)Elastase Proenzyme with variant Cleavage Domain 54, with Amino Acid 102possible polymorphisms at positions 10 (Q or H); 44 (W or R); 59 (M orV); 220 (V or L); and 243 (Q or R) (numbering refers to position incontext of preproprotein) Elastase Proenzyme with variant CleavageDomain 55, with Amino Acid 103 possible polymorphisms at positions 10 (Qor H); 44 (W or R); 59 (M or V); 220 (V or L); and 243 (Q or R)(numbering refers to position in context of preproprotein) Wild TypeElastase + AlaArg Cleavage Variant, with Amino Acid 104 possiblepolymorphisms at positions 44 (W or R); 59 (M or V); 220 (V or L); and243 (Q or R) (numbering refers to position in context of preproprotein)Wild Type Elastase + Arg Cleavage Variant, with possible Amino Acid 105polymorphisms at positions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (Vor L); and 243 (Q or R) (numbering refers to position in context ofpreproprotein) Mature human elastase I cleavage variant lacking firstfour Amino Acid 106 amino acids, with possible polymorphisms atpositions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (V or L); and 243(Q or R) (numbering refers to position in context of preproprotein)Mature human elastase I cleavage variant lacking first six Amino Acid107 amino acids, with possible polymorphisms at positions 44 (W or R);59 (M or V); 220 (V or L); and 243 (Q or R) (numbering refers toposition in context of preproprotein) Mature human elastase I cleavagevariant lacking first nine Amino Acid 108 amino acids, with possiblepolymorphisms at positions 44 (W or R); 59 (M or V); 220 (V or L); and243 (Q or R) (numbering refers to position in context of preproprotein)Nucleic acid sequence of FIG. 1A Nucleic Acid 109 Amino acid sequence ofFIG. 1A Amino Acid 110 Nucleic acid sequence of FIG. 1B Nucleic Acid 111Amino acid sequence of FIG. 1B Amino Acid 112 Nucleic acid sequence ofFIG. 13 Nucleic Acid 113 Amino acid sequence of FIG. 14 Amino Acid 114Cleavage domain sequence of trypsin-activated pPROT101- Amino Acid 11524-V Cleavage domain sequence of auto-activated Amino Acid 116pPROT101-42-V Cleavage domain sequence of auto-activated Amino Acid 117pPROT101-49-V Cleavage domain sequence of auto-activated Amino Acid 118pPROT101-55L-V Consensus elastase recognition sequence 5 (Positions P3-Amino Acid 119 P2-P1) Consensus sequence 1 for the propeptide portion ofthe Amino Acid 120 cleavage domain; corresponds to residues P5, P4, P3,P2, and P1 Consensus sequence 2 for the activation peptide; Amino Acid121 corresponds to residues P10 through P1 Proelastase consensussequence 1 for residues P10 through Amino Acid 122 P3′ Consensuscleavage domain sequence 2; corresponds to Amino Acid 123 residues P5,P4, P3, P2, P1, P′1, P′2, and P′3 Consensus elastase recognitionsequence 6 (Positions P3- Amino Acid 124 P2-P1) Consensus cleavagedomain sequence 3; corresponds to Amino Acid 125 residues P5, P4, P3,P2, P1, P′1, P′2, and P′3

There are at least 5 confirmed polymorphisms in human type I elastaseprotein, at positions 10 (Q or H); 44 (W or R); 59 (M or V); 220 (V orL); and 243 (Q or R). The full-length (preproelastase) protein is 258amino acids in length. The first 8 amino acids correspond to the signalor “pre” peptide sequence that is cleaved off to generate an inactiveproprotein, and a further “pro” peptide sequence (comprising orconsisting of 10 amino acids corresponding to an activation peptide) arecleaved to generate the active, mature protein. Thus, the polymorphismat position 10 is present in the proprotein but not in the matureprotein.

Accordingly, in Table 2 below are presented all possible combinations ofthe 5 polymorphisms of human type I elastase. The present inventionprovides preproelastase and proelastase proteins (including but notlimited to the variant preproelastase and proelastase proteins describedherein), such as the proteins exemplified in embodiments 1-39 and 68-69or the proteins obtained or obtainable by the method of any one ofembodiments 89-224, 261-276, and 347-373 in Section 8, comprising theany of the combinations of polymorphisms set forth in Table 2 below.

TABLE 2 Variants of human type I immature elastase (pre-pro) and proelastase proteins. The position numbering refers to the position in thecontext of the preproprotein of native human type I elastase. Position10 44 59 220 243 Embodiment Q or H W or R M or V V or L Q or R 1. Q W MV Q 2. Q W M V R 3. Q W M L Q 4. Q W V V Q 5. Q R M V Q 6. Q W M L R 7.Q W V L Q 8. Q R V V Q 9. Q W V V R 10. Q R M L Q 11. Q R M V R 12. Q RV L Q 13. Q R V V R 14. Q R M L R 15. Q W V L R 16. Q R V L R 17. H W MV Q 18. H W M V R 19. H W M L Q 20. H W V V Q 21. H R M V Q 22. H W M LR 23. H W V L Q 24. H R V V Q 25. H W V V R 26. H R M L Q 27. H R M V R28. H R V L Q 29. H R V V R 30. H R M L R 31. H W V L R 32. H R V L R

Moreover, in Table 3 below are presented all possible combinations ofthe 4 polymorphisms of human type I elastase that may be present in amature elastase protein. The present invention provides mature elastaseproteins (including but not limited to the variant mature elastaseproteins described herein), such as the mature elastase proteinsobtained or obtainable by the method of any one of embodiments 89-224,261-276, and 347-373 in Section 8, comprising the any of thecombinations of polymorphisms set forth in Table 3 below.

TABLE 3 Variants of mature human type I elastase proteins. The positionnumbering refers to the position in the context of the preproprotein ofnative human type I elastase. Position 44 59 220 243 Embodiment W or R Mor V V or L Q or R 1. W M V Q 2. W M V R 3. W M L Q 4. W V V Q 5. R M VQ 6. W M L R 7. W V L Q 8. R V V Q 9. W V V R 10. R M L Q 11. R M V R12. R V L Q 13. R V V R 14. R M L R 15. W V L R 16. R V L R

The mature type I elastases of the invention can be purified, forexample for use in pharmaceutical compositions. In specific embodiments,the elastases are at least 70%, 80%, 90%, 95%, 98% or 99% pure.

The mature type I elastases of the invention preferably have a specificactivity of greater than 1, greater than 5, greater than 10, greaterthan 20, greater than 25, or greater than 30 U/mg of protein, asdetermined by measuring the rate of hydrolysis of the small peptidesubstrate N-succinyl-Ala-Ala-Ala-pNitroanilide (SLAP), which iscatalyzed by the addition of elastase. One unit of activity is definedas the amount of elastase that catalyzes the hydrolysis of 1 micromoleof substrate per minute at 30° C. and specific activity is defined asactivity per mg of elastase protein (U/mg). Preferably, a mature humantype I elastase has a specific activity within a range in which thelower limit is 1, 2, 3, 4, 5, 7, 10, 15 or 20 U/mg protein and in whichthe upper limit is, independently, 5, 10, 15, 20, 25, 30, 35, 40 or 50U/mg protein. In exemplary embodiments, the specific activity is in therange of from 1 to 40 U/mg of protein, from 1 to 5 U/mg of protein, from2 to 10 U/mg of protein, from 4 to 15 U/mg of protein, from 5 to 30 U/mgof protein, from 10 to 20 U/mg of protein, from 20 to 40 U/mg ofprotein, or any range whose upper and lower limits are selected from anyof the foregoing values. A mature porcine type I elastase preferably hasa specific activity within a range in which the lower limit is 1, 2, 3,4, 5, 7, 10, 15, 20, 30, 40, 50, 60, or 75 U/mg protein and in which theupper limit is, independently, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60,75, 90, 95 or 100 U/mg protein. In exemplary embodiments, the specificactivity of the porcine elastase is in the range of from 10 to 50 U/mgof protein, from 20 to 60 U/mg of protein, from 30 to 75 U/mg ofprotein, from 30 to 40 U/mg of protein, from 20 to 35 U/mg of protein,from 50 to 95 U/mg of protein, from 25 to 100 U/mg of protein, or anyrange whose upper and lower limits are selected from any of theforegoing values.

Accordingly, certain aspects of the present invention relate tocompositions, such as pharmaceutical compositions, elastase formulationsand unit dosages, such as those exemplified in embodiments 277-314, 346,386, and 413-420 or those obtained or obtainable by the method of anyone of embodiments 261-276 and 374-385 in Section 8 below.

In certain embodiments, the compositions of the invention comprisetrypsin-activated elastase proteins, e.g., trypsin activated proteinsmade by any of the methods disclosed herein. In other embodiments, thecompositions comprise autoactivated elastase proteins, e.g.,autoactivated elastase proteins made by any of the methods disclosedherein. In certain aspects, a composition of the invention ischaracterized by at least one, at least two, at least three, at leastfour, at least five, at least six or at least seven of the followingproperties: (a) the composition is free of trypsin; (b) the compositionis substantially free of trypsin; (c) the composition is free of anyprotein consisting of SEQ ID NOS:70 and 71; (d) the composition issubstantially free of any protein consisting of SEQ ID NOS:2 and 3; (e)the composition is free of bacterial proteins; (f) the composition issubstantially free of bacterial proteins; (g) the composition is free ofmammalian proteins other than said mature elastase protein; (h) thecomposition is substantially free of mammalian proteins other than saidmature elastase protein; (i) the composition is free or substantiallyfree of one, two, three or all four proteins consisting of SEQ ID NO:85,86, 94 and 95; (j) the composition is free or substantially free of one,two, or all three proteins consisting of SEQ ID NO:106, 107 and 108; (k)the composition contains pharmaceutically acceptable levels ofendotoxins (e.g., 10 EU/mg elastase or less, or 5 EU/mg elastase orless); (1) the mature elastase protein in the composition ischaracterized by a specific activity of 1 to 40 U/mg of protein or anyother range of specific activity disclosed herein; (m) the trypsinactivity in said composition corresponds to less than 4 ng per 1 mg ofmature elastase protein or any other range of trypsin activity disclosedherein; (n) the composition comprises polysorbate-80; (o) thecomposition comprises dextran; (p) the composition comprises sodiumions, potassium ions, phosphate ions, chloride ions and polysorbate-80;(q) the composition comprises sodium ions, potassium ions, phosphateions, chloride ions and dextran; (r) the composition comprises sodiumions, potassium ions, phosphate ions, chloride ions, polysorbate-80, anddextran; (s) the mature elastase protein in said composition displays anamount of stability disclosed herein, e.g., maintains 60% to 100% of itsspecific activity after at least a week of storage at 4° C., after atleast a month of storage at 4° C., after at least two months of storageat 4° C., after at least three months of storage at 4° C., or after atleast month six months of storage at 4° C.; and (t) the compositioncomprises a unit dosage of 0.0033 mg to 200 mg of said mature elastaseprotein, or any other unit dosage of mature elastase protein disclosedherein.

In certain aspects, the composition is characterized by at least threecharacteristics, at least four characteristics or five characteristicsindependently selected from the following groups (i) through (v):

-   -   (i) (a), (b) or (m)    -   (ii) (e) or (f)    -   (iii) (g) or (h)    -   (iv) (k)    -   (v) (l)

In specific embodiments, two of said at least three or at least saidfour characteristics are selected from groups (i) and (iv) or (v). Inother embodiments, three of at least said four characteristics areselected from groups (i), (iv) and (v).

In specific embodiments, the present invention provides a composition,including but not limited to a pharmaceutical composition, elastaseformulation or unit dosage, comprising (i) a therapeutically effectiveamount of human type I elastase that is free of trypsin and (ii) apharmaceutically acceptable carrier. Alternatively, the presentinvention provides a composition comprising (i) a therapeuticallyeffective amount of human type I elastase that is substantially free oftrypsin and (ii) a pharmaceutically acceptable carrier. In specificembodiments, the human type I elastase and/or the composition comprisesless than 5 ng/ml of trypsin activity, less than 4 ng/ml of trypsinactivity, less than 3 ng/ml of trypsin activity, less than 2 ng/ml oftrypsin activity, or less than 1.56 ng/ml of trypsin activity. In theforegoing examples, the ng/ml trypsin activity can be assayed in aliquid human type I elastase composition or preparation containing 1mg/ml human type I elastase protein. Thus, the trypsin activities mayalso be described in terms of milligrams of elastase protein, forexample, less than 3 ng trypsin activity/mg elastase protein, less than1.56 ng trypsin activity/mg elastase protein, etc. The present inventionfurther provides a composition comprising (i) a therapeuticallyeffective amount of human type I elastase and (ii) a pharmaceuticallyacceptable carrier, wherein the composition comprises less than 5 ng oftrypsin activity per mg of elastase, less than 4 ng trypsin activity permg of elastase, less than 3 ng of trypsin activity per mg of elastase,less than 2 ng of trypsin activity per mg of elastase, or less than 1.56ng of trypsin activity per mg of elastase.

The present invention further provides methods of improving the qualityof mature elastase proteins produced by trypsin activation methods(e.g., the methods of embodiment 145 in Section 8 below), comprisingpurifying the mature elastase protein on a Macro-Prep High S Resincolumn. It was found by the present inventors that mature elastaseproteins purified on a Macro-Prep High S Resin column yields elastasecompositions with trypsin activity levels corresponding 20-25 ng trypsinactivity/mg elastase protein, as compared to purification on a Poros(Poly (Styrene-Divinylbenzene)) column which yields elastasecompositions with trypsin activity levels corresponding to approximately1000 ng trypsin activity/mg elastase protein.

The present invention further provides elastase compositions comprisingmature elastase proteins produced by purifying trypsin-activatedelastase proteins on a Macro-Prep High S Resin column. The Macro-PrepHigh S Resin is available from Biorad (Hercules, Calif.), according towhom a column of rigid methacrylate supports with little shrinkage andswelling. Other similar cation exchange columns of the same class and/orwith the same bead size (around 50 μm) may be used, such as othermethacrylate columns, may suitably be used.

Other aspects of the present invention relate to compositions, includingbut not limited to pharmaceutical compositions, elastase formulations orunit dosages, comprising porcine type I pancreatic elastase. In specificembodiments, the present invention provides a composition comprising (i)a therapeutically effective amount of porcine type I pancreatic elastasethat is free of trypsin and (ii) a pharmaceutically acceptable carrier.Alternatively, the present invention provides a composition comprising(i) a therapeutically effective amount of porcine type I pancreaticelastase that is substantially free of trypsin and (ii) apharmaceutically acceptable carrier. In specific embodiments, theporcine type I pancreatic elastase and/or the composition comprises lessthan 100 ng/ml of trypsin activity, less than 75 ng/ml of trypsinactivity, less than 50 ng/ml of trypsin activity, less than 25 ng/ml oftrypsin activity, less than 15 ng/ml of trypsin activity, less than 10ng/ml of trypsin activity, less than 5 ng/ml of trypsin activity, lessthan 4 ng/ml of trypsin activity, less than 3 ng/ml of trypsin activity,less than 2 ng/ml of trypsin activity, or less than 1.56 ng/ml oftrypsin activity. In the foregoing examples, the ng/ml trypsin activitycan be assayed in a liquid porcine type I pancreatic elastasecomposition or preparation containing 1 mg/ml porcine type I pancreaticelastase. Thus, the trypsin activities may also be described in terms ofmilligrams of elastase protein, for example, less than 25 ng trypsinactivity/mg elastase protein, less than 5 ng trypsin activity/mgelastase protein, etc. The present invention further provides acomposition comprising (i) a therapeutically effective amount of porcinetype I elastase and (ii) a pharmaceutically acceptable carrier, whereinthe composition comprises than 100 ng of trypsin activity per mg ofelastase, less than 75 ng trypsin activity per mg of elastase, less than50 ng of trypsin activity per mg of elastase, less than 25 ng of trypsinactivity per mg of elastase, less than 15 ng of trypsin activity per mgof elastase, less than 10 ng or trypsin activity per mg of elastase,less than 5 ng of trypsin activity per mg of elastase, less than 4 ngtrypsin activity per mg of elastase, less than 3 ng of trypsin activityper mg of elastase, less than 2 ng of trypsin activity per mg ofelastase, or less than 1.56 ng of trypsin activity per mg of elastase.

In other embodiments, the present invention provides compositions ofelastase proteins, such as mature elastase proteins, including but notlimited to pharmaceutical compositions, elastase formulations or unitdosages, that are free of N-terminal variants corresponding to one, two,three or all four of SEQ ID NOS: 70, 71, 104, 105. In certainembodiments, the present invention provides a pharmaceutical compositioncomprising (i) a therapeutically effective amount of mature human type Ielastase (ii) a pharmaceutically acceptable carrier, whichpharmaceutical composition is free of any protein with SEQ ID NOS:70,71, 104, 105.

In other embodiments, the present invention provides a composition,including but not limited to a pharmaceutical composition, an elastaseformulation or unit dosage, comprising (i) a therapeutically effectiveamount of human type I elastase that is free or substantially free ofvariant proteins containing specific additional amino acids on theN-terminal end of the mature elastase protein (SEQ ID NOS: 37, 38, 70,71, 94, 95, 104, 105) and (ii) a pharmaceutically acceptable carrier. Inother embodiments, the present invention provides a compositioncomprising (i) a therapeutically effective amount of human type Ielastase that is free or substantially free of variant proteins lackingN-terminal amino acids of the mature elastase protein (SEQ ID NOS: 2, 3,37, 38, 70, 71, 85, 86, 94, 95, 104, 105, 106, 107, 108) and (ii) apharmaceutically acceptable carrier. In specific embodiments, the humantype I elastase and/or the composition comprises less than 25%N-terminal variants, less than 20% N-terminal variants, less than 15%N-terminal variants, less than 10% N-terminal variants, less than 5%N-terminal variants, less than 4% N-terminal variants, less than 3%N-terminal variants, less than 2% N-terminal variants, less than 1%N-terminal variants, less than 0.5% N-terminal variants, 0% N-terminalvariants, or comprises N-terminal variants in an amount ranging betweenany two of the foregoing percentages (e.g., 2%-25% N-terminal variants,0.5%-10% N-terminal variants, 5%-15% N-terminal variants, 0%-2%N-terminal variants, etc.). As used herein, the term “less than X %N-terminal variants” refers to the amount of N-terminal variants as apercentage of total elastase protein.

In other embodiments, the present invention provides a composition,including but not limited to a pharmaceutical composition, an elastaseformulation or unit dosage, comprising (i) a therapeutically effectiveamount of mature human type I elastase (ii) a pharmaceuticallyacceptable carrier, which pharmaceutical composition is substantiallyfree of bacterial proteins and/or is substantially free of mammalianproteins other than said mature human type I elastase. In specificembodiments, the human type I elastase and/or the composition comprisesless than 25% bacterial proteins and/or mammalian proteins other thansaid mature human type I elastase, less than 20% bacterial proteinsand/or mammalian proteins other than said mature human type I elastase,less than 15% bacterial proteins and/or mammalian proteins other thansaid mature human type I elastase, less than 10% bacterial proteinsand/or mammalian proteins other than said mature human type I elastase,less than 5% bacterial proteins and/or mammalian proteins other thansaid mature human type I elastase, less than 4% bacterial proteinsand/or mammalian proteins other than said mature human type I elastase,less than 3% bacterial proteins and/or mammalian proteins other thansaid mature human type I elastase, less than 2% bacterial proteinsand/or mammalian proteins other than said mature human type I elastase,less than 1% bacterial proteins and/or mammalian proteins other thansaid mature human type I elastase, less than 0.5% bacterial proteinsand/or mammalian proteins other than said mature human type I elastase,0% bacterial proteins and/or mammalian proteins other than said maturehuman type I elastase, or comprises bacterial proteins and/or mammalianproteins other than said mature human type I elastase in an amountranging between any two of the foregoing percentages (e.g., 2%-25%bacterial proteins and/or mammalian proteins other than said maturehuman type I elastase, 0.5%-10% bacterial proteins and/or mammalianproteins other than said mature human type I elastase, 5%-15% bacterialproteins and/or mammalian proteins other than said mature human type Ielastase, 0%-2% bacterial proteins and/or mammalian proteins other thansaid mature human type I elastase, etc.). As used herein, the term “lessthan X % bacterial proteins and/or mammalian proteins other than saidmature human type I elastase” refers to the amount of such proteins as apercentage of total protein in an elastase preparation or in saidcomposition. In certain embodiments, the elastase represents at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5% or at least 99.8% of the total protein in such compositions orpreparations.

Methods for the treatment and prevention of diseases of biologicalconduits, comprising administration of compositions (e.g.,pharmaceutical compositions, elastase formulations or unit dosages)comprising a purified mature human type I elastase of the invention to apatient in need thereof, are also provided.

Further provided are vectors comprising nucleic acids encoding any ofthe elastase proteins of the invention (“nucleic acids of theinvention”), host cells engineered to express the nucleic acids of theinvention. In specific embodiments, the vectors further comprise anucleotide sequence which regulates the expression of the elastaseprotein. For example, the nucleotide sequence encoding the protein ofthe invention can be operably linked to a methanol-inducible promoter.Other suitable promoters are exemplified in Section 5.3 below.

In a specific embodiment, the present invention provides a vectorcomprising a nucleotide sequence encoding an open reading frame, theopen reading frame comprising a yeast α-factor signal peptide or a typeI elastase signal peptide (e.g., porcine elastase signal peptide)operably linked to a human type I elastase proprotein sequence.Optionally, the vector further comprises a methanol-inducible promoteroperably linked to the open reading frame.

Host cells comprising the nucleic acids and vectors of the invention arealso provided. In certain embodiments, the vector or nucleic acid isintegrated into the host cell genome; in other embodiments, the vectoror nucleic acid is extrachromosomal. A preferred host cell is a Pichiapastoris cell. Other suitable host cells are exemplified in Section 5.3below.

In a specific embodiment, the present invention provides a Pichiapastoris host cell genetically engineered to encode an open readingframe comprising a yeast α-factor signal peptide or a porcine elastasesignal peptide operably linked to a human type I elastase proenzymesequence. Optionally, the open reading frame is under the control of amethanol-inducible promoter.

The present invention further provides methods for producing an immatureor mature elastase protein of the invention comprising culturing a hostcell engineered to express a nucleic acid of the invention underconditions in which the proelastase protein is produced. In certainembodiments, the mature elastase protein is also produced.

Preferred culture conditions for producing the proelastase and matureproteins of the invention, particularly for the host cell Pichiapastoris, include a period of growth at a low pH. Typically, the low pHis 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or any range between anypair of the foregoing values. In specific embodiments, the low pH is apH ranging from 2.0 to 6.0, from 2.0 to 5.0, from 3.0 to 6.0, from 3.0to 5.0, from 4.0 to 6.0, or from 3.0 to 4.0. At the end of the cultureperiod, the pH of the culture can be raised, preferably to a pH of 7.0,7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0 or a pH ranging between anytwo of the foregoing values for the purpose of separating or removingthe activation sequence from the mature elastase protein. In specificembodiments, the pH of the culture is raised to a pH ranging from 7.5 to10.0 or 8.0 to 9.0 and most preferably to a pH of 8.0.

Where the expression of a proelastase protein of the invention is underthe control of a methanol-inducible promoter, conditions for producingan immature or mature elastase protein of the invention may alsocomprise a period of methanol induction.

The elastase production methods of the invention may further comprisethe step of recovering the protein expressed by the host cell. Incertain instances, the protein recovered is a proelastase comprising theactivation sequence. In other instances, the protein recovered is amature elastase lacking the activation sequence. Under certainconditions, both proelastase and mature elastase proteins are recovered.

Preferably, particularly where it is desired to circumventauto-activation of an auto-activated proelastase, culture conditions forproelastase expression may comprise a period of growth and induction atlow pH. Typically, the low pH is 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,or 6.0, or any range between any pair of the foregoing values. Inspecific embodiments, the low pH is a pH ranging from 2.0 to 3.0, from4.0 to 5.0, from 5.0 to 6.0, or from 6.0 to 7.0. In particularembodiments, the pH ranges from 4.0 to 6.0 and is most preferably a pHof 5.5.

Preferably, particularly where it is desired to circumventauto-activation of an auto-activated proelastase, culture conditions forproelastase expression comprise a period of growth and induction insodium citrate, sodium succinate, or sodium acetate. In specificembodiments, a concentration of 5-50 mM, 7.5-100 mM, 10-15 mM, 50-200mM, 75-175 mM, 100-150 mM, 75-125 mM, or of any range whose upper andlower limits are selected from any of the foregoing values (e.g., 50-75mM, 75-100 mM, etc.) is used. In a preferred embodiment, the sodiumcitrate, sodium succinate, or sodium acetate concentration is 90-110 mMand most preferably is 100 mM.

Additionally, particularly where it is desired to circumventauto-activation of an auto-activated proelastase or auto-degradation bymature elastase, culture conditions for expression of an immatureelastase protein may comprise a period of growth and induction at thelower end of the temperature range suitable for the host cell inquestion. For example, where the host cell is a Pichia pastoris hostcell, the preferred range is about 22-28° C. In a specific embodiment,the Pichia pastoris host cell is cultured at 28° C.

Additionally, particularly where it is desired to circumvent proteindegradation by host cell proteases, culture conditions for expression ofan immature elastase protein may comprise a period of growth andinduction at the lower end of the temperature range suitable for thehost cell in question. For example, where the host cell is a Pichiapastoris host cell, the preferred range is about 22-28° C. In a specificembodiment, the Pichia pastoris host cell is cultured at 28° C.

The activation of an auto-activated proelastase protein of the inventionmay be initiated by the addition of extrinsic elastase in a small(catalytic) amount. In certain embodiments, a catalytic amount ofextrinsic elastase represents less than 10%, less than 5%, less than 2%,less than 1%, less than 0.5% or less than 0.1%, on either a molar ormolecular weight basis, of the elastase in the sample to which thecatalytic elastase is added.

Alternatively or concurrently, the auto-activated proelastase may besubjected to pH 7-11 (most preferably pH 8), upon which theauto-activated proelastase activation peptide is removed withoutrequiring trypsin and resulting in mature, active elastase. In specificembodiments, Tris base is added to a concentration of 50-200 mM, 75-175mM, 100-150 mM, 75-125 mM, or any range whose upper and lower limits areselected from any of the foregoing values (e.g., 50-75 mM, 75-100 mM,etc.) during the activation step. In a preferred embodiment, Tris baseis added to a concentration 90-110 mM, most preferably 100 mM. The pH ofthe Tris base is preferably 7-11; in specific embodiments, the Tris baseis at a pH of 7.0-11.0, 7-9, 7.5 to 9.5, 7.5 to 10, 8-10, 8-9, or anyrange whose upper and lower limits are selected from any of theforegoing values. In a preferred embodiment, the Tris base is at a pH of7.5-8.5, most preferably 8.0.

Expression of an immature elastase sequence can in some instances yielda mixture of proelastase proteins and mature elastase proteins, as wellas N-terminal variant elastase proteins. Thus, the present inventionprovides a composition comprising at least two of (1) a proelastaseprotein, (2) a mature elastase protein, and (3) N-terminal variantelastase proteins.

Once a mature elastase is produced, it can be lyophilized, for examplefor pharmaceutical formulations. In an exemplary embodiment, the presentinvention provides methods of isolating a lyophilized mature type Ielastase comprising steps:

(a) culturing a host cell, such as a Pichia pastoris host cell,engineered to express a nucleic acid molecule encoding a preproelastaseopen reading frame under conditions in which the open reading frame isexpressed, wherein, in a specific embodiment, said open reading framecomprises nucleotide sequences encoding, in a 5′ to 3′ direction (i) asignal peptide, e.g., a single peptide operable in Pichia pastoris; (ii)an activation sequence comprising an elastase recognition sequence; and(iii) the sequence of a mature type I elastase protein, therebyproducing a proelastase protein;

(b) subjecting the proelastase protein to autoactivation conditions,thereby producing a mature type I elastase, wherein the autoactivationconditions include, one or a combination of the following:

-   -   (i) changing the pH of a solution (which can be a cell culture        supernatant) containing the proelastase protein, e.g., to a pH        of 6.5-11, preferably 8-9;    -   (ii) purifying the proelastase protein, for example, by ion        exchange chromatography, and subjecting the solution extended        conversion to remove N-terminal variants, thereby producing        mature human type I elastase;    -   (iii) concentrating the proelastase protein (e.g., 2-fold,        3-fold, 5-fold, 8-fold, 10-fold, 12-fold, or a range in which        the upper and lower limits are independently selected from the        foregoing levels of concentrations);    -   (iv) subjecting the proelastase protein to increased temperature        (e.g., 29° C., 30° C., 32° C., 35° C., 40° C., 45° C., or 40°        C., or a range in which the upper and lower limits are        independently selected from the foregoing temperatures);    -   (v) purifying the proelastase protein (e.g., using Macro-Prep        High S Resin) from a cell culture supernatant and incubating a        solution comprising the purified proelastase protein at ambient        temperatures (e.g., 22° C. to 26° C.) for a period of at least        one day (e.g., one day, two days, three days, four days five        days, or six days, a range of days in which the upper and lower        limits are independently selected from the foregoing values)        (this is influenced by the presence of citrate/acetate,        concentration, temperature, and pH in the solution, and can        readily be determined by one of skill in the art).

(c) optionally, purifying the mature human type I elastase, e.g., ionexchange chromatography step for polish chromatography; and

(d) lyophilizing the mature type I elastase, thereby isolating alyophilized mature human type I elastase. The mature type I elastase ispreferably a human type I elastase. In certain aspects, the lyophilizedmature type I elastase is preferably more than 95% pure; in specificembodiments, the lyophilized mature type I elastase is more than 98% ormore than 99% pure.

The mature elastase proteins of the invention can be formulated intopharmaceutical compositions. Thus, in exemplary embodiments, the presentinvention provides a method of generating a pharmaceutical compositioncomprising a mature human type I elastase, said method comprising (i)isolating a lyophilized mature human type I elastase according to themethods described above; and (ii) reconstituting the lyophilized maturehuman type I elastase in a pharmaceutically acceptable carrier. Themature human type I elastases of the invention preferably have aspecific activity of greater than 1, greater than 5, greater than 10,greater than 20, greater than 25, or greater than 30 U/mg of protein, asdetermined by measuring the rate of hydrolysis of the small peptidesubstrate N-succinyl-Ala-Ala-Ala-pNitroanilide (SLAP), which iscatalyzed by the addition of elastase. One unit of activity is definedas the amount of elastase that catalyzes the hydrolysis of 1 micromoleof substrate per minute at 30° C. and specific activity is defined asactivity per mg of elastase protein (U/mg). Preferably, a mature humantype I elastase of the invention has a specific activity within a rangein which the lower limit is 1, 2, 3, 4, 5, 7, 10, 15 or 20 U/mg proteinand in which the upper limit is, independently, 5, 10, 15, 20, 25, 30,35, 40 or 50 U/mg protein. In exemplary embodiments, the specificactivity is in the range of 1-40 U/mg of protein, 1-5 U/mg protein, 2-10U/mg protein, 4-15 U/mg protein, 5-30 U/mg of protein, 10-20 U/mg ofprotein, 20-40 U/mg of protein, or any range whose upper and lowerlimits are selected from any of the foregoing values (e.g., 1-10 U/mg,5-40 U/mg, etc.).

The pharmaceutical compositions of the invention are preferably stable.In specific embodiments, a pharmaceutical composition (for example apharmaceutical composition prepared by lyophilization and reconstitutionas described above) maintains at least 50%, more preferably at least60%, and most preferably at least 70% of its specific activity after aweek of storage at 4° C. In specific embodiments, the pharmaceuticalcomposition maintains at least 75%, at least 80%, at least 85% or atleast 95% of its specific activity after reconstitution and a week ofstorage at 4° C.

This invention also provides proteins comprising a type I elastaseproprotein amino acid sequence containing an elastase cleavage domainsequence. Other cleavage domains that can be used in this invention areany of the sequences described by the consensus cleavage domain sequence(SEQ ID NO:74) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈, where Xaa₁=P5,Xaa₂=P4, Xaa₃=P3, Xaa₄=P2, Xaa₅=P1, Xaa₆=P′1, Xaa₇=P′2, and Xaa₈=P′3,where:

-   -   Xaa₁ is glutamate, histidine, proline, glycine, asparagine,        lysine, or alanine, or, optionally, an analog thereof;    -   Xaa₂ is threonine, alanine, proline or histidine or, optionally,        an analog thereof;    -   Xaa₃ is alanine, leucine, isoleucine, methionine, lysine,        asparagine or valine, or, optionally, an analog thereof, but is        preferably not glycine or proline;    -   Xaa₄ is proline, alanine, leucine, isoleucine, glycine, valine,        or threonine, or, optionally, an analog thereof;    -   Xaa₅ is alanine, leucine, valine, isoleucine, or serine but not        glycine, tyrosine, phenylalanine, proline, arginine, glutamate,        or lysine, or, optionally, an analog thereof;    -   Xaa₆ is alanine, leucine, valine, isoleucine or serine, or,        optionally, an analog thereof;    -   Xaa₇ is glycine, alanine, or valine, or, optionally, an analog        thereof; and    -   Xaa₈ is valine, threonine, phenylalanine, tyrosine, or        tryptophan, or, optionally, an analog thereof.

This invention also provides proteins comprising a type I elastaseproprotein amino acid sequence containing an elastase cleavage domainsequence. Other cleavage domains that can be used in this invention areany of the sequences described by the consensus cleavage domain sequence(SEQ ID NO:123) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈, where Xaa₁=P5,Xaa₂=P4, Xaa₃=P3, Xaa₄=P2, Xaa₅=P1, Xaa₆=P′1, Xaa₇=P′2, and Xaa₈=P′3,where:

-   -   Xaa₁ is glutamate, histidine, proline, glycine, asparagine,        lysine, or alanine, or, optionally, an analog thereof;    -   Xaa₂ is threonine, alanine, proline or histidine or, optionally,        an analog thereof;    -   Xaa₃ is alanine, leucine, isoleucine, methionine, lysine,        asparagine or valine, or, optionally, an analog thereof, but is        preferably not glycine or proline;    -   Xaa₄ is proline, alanine, leucine, isoleucine, glycine, valine,        or threonine, or, optionally, an analog thereof;    -   Xaa₅ is alanine, leucine, valine, isoleucine, or serine but not        glycine, tyrosine, phenylalanine, proline, arginine, glutamate,        or lysine, or, optionally, an analog thereof;    -   Xaa₆ is alanine, leucine, valine, isoleucine or serine, or,        optionally, an analog thereof;    -   Xaa₇ is glycine, alanine, or valine, or, optionally, an analog        thereof; and    -   Xaa₈ is glycine, valine, threonine, phenylalanine, tyrosine, or        tryptophan, or, optionally, an analog thereof.

This invention also provides proteins comprising a type I elastaseproprotein amino acid sequence containing an elastase cleavage domainsequence. Other cleavage domains that can be used in this invention areany of the sequences described by the consensus cleavage domain sequence(SEQ ID NO:125) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈, where Xaa₁=P5,Xaa₂=P4, Xaa₃=P3, Xaa₄=P2, Xaa₅=P1, Xaa₆=P′1, Xaa₇=P′2, and Xaa₈=P′3,where:

-   -   Xaa₁ is glutamate, histidine, proline, glycine, asparagine,        lysine, or alanine, or, optionally, an analog thereof;    -   Xaa₂ is threonine, alanine, proline or histidine or, optionally,        an analog thereof;    -   Xaa₃ is alanine, leucine, isoleucine, methionine, lysine,        asparagine, threonine or valine, or, optionally, an analog        thereof, but is preferably not glycine or proline;    -   Xaa₄ is proline, alanine, leucine, isoleucine, glycine, valine,        asparagine, or threonine, or, optionally, an analog thereof;    -   Xaa₅ is alanine, leucine, valine, isoleucine, or serine but not        glycine, tyrosine, phenylalanine, proline, arginine, glutamate,        asparagine, or lysine, or, optionally, an analog thereof;    -   Xaa₆ is alanine, leucine, valine, isoleucine or serine, or,        optionally, an analog thereof;    -   Xaa₇ is glycine, alanine, or valine, or, optionally, an analog        thereof; and    -   Xaa₈ is glycine, valine, threonine, phenylalanine, tyrosine, or        tryptophan, or, optionally, an analog thereof.

In certain embodiments relating SEQ ID NO:74, SEQ ID NO:123, or SEQ IDNO:125, Xaa₁ is histidine and/or Xaa₄ is proline and/or Xaa₅ is alanine.

This invention also provides a method of isolating a mature human type Ielastase comprising: (a) culturing, under culturing conditions, a hostcell comprising a nucleotide sequence which encodes a proproteincomprising (i) an activation sequence comprising a trypsin recognitionsequence operably linked to (ii) the amino acid sequence of a proteinhaving elastase activity under said culturing conditions, wherein saidculturing conditions comprise a period of growth or induction at pH of 2to 6; (b) recovering the expressed proprotein; (c) contacting therecovered protein with a catalytic amount of trypsin under pH conditionsin which the trypsin is active; and (d) isolating mature human type Ielastase. In this method the mature human type I elastase may consistessentially of SEQ ID NO: 1, 4, 5, 84, or 87. In certain embodiments,the conditions may comprise (a) a period of growth or induction at pH of4 to 6; (b) a period of growth or induction at 22° C. to 28° C.; or (c)sodium citrate, sodium succinate, or sodium acetate concentrations ofabout 50 mM to about 200 mM or a sodium citrate concentration is 90 mMto about 110 mM in the culture media of said host cells.

It should be noted that the indefinite articles “a” and “an” and thedefinite article “the” are used in the present application, as is commonin patent applications, to mean one or more unless the context clearlydictates otherwise. Further, the term “or” is used in the presentapplication, as is common in patent applications, to mean thedisjunctive “or” or the conjunctive “and.”

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like that has been included in this specification issolely for the purpose of providing a context for the present invention.It is not to be taken as an admission that any or all of these mattersform part of the prior art base or were common general knowledge in thefield relevant to the present invention as it existed anywhere beforethe priority date of this application.

The features and advantages of the invention will become furtherapparent from the following detailed description of embodiments thereof.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B: FIG. 1A shows the synthetic (i.e., recombinant) humanELA-1.2A sequence (SEQ ID NO:130). The recombinant human elastase-1(i.e., human type I pancreatic elastase) sequence contains a 750-basepair coding region. Selected restriction enzyme sites are underlined.Base substitutions are in double underlined, bolded text and the codonscontaining them are double underlined. Stop codons are shaded in gray(but not underlined). The propeptide sequence is italicized. The codingregion results in a 250-amino acid protein (SEQ ID NO:82). Aftercleavage of the 10-amino acid propeptide, the resulting mature enzyme is240 amino acids. FIG. 1B shows the pPROT24 translational fusion region.The translational fusion between the vector and the ELA-1 coding regionis depicted. The PCR amplification of the ELA-1 sequence provided forthe incorporation of the Kex2 and STE13 signal cleavage domains to yielda secreted product with an expected N-terminus of the first amino acid(in bold text) of the activation sequence (italicized). FIG. 1Bdiscloses SEQ ID NOS:111 and 112, respectively, in order of appearance.

FIG. 2: A N-terminal to C-terminal schematic of the (overlapping) corecomponents of the elastase proteins of the invention, wherein thenumbered components depict: (1) signal sequence (vertical stripes); (2)optional propeptide/spacer sequence (bricks); (3) elastase propeptide(diagonal stripes, gray fill and diamond pattern combined); (4)activation peptide (gray fill and diamond pattern combined); (5)recognition sequence (diamond pattern); (6) cleavage domain (gray fill,diamond pattern and the left portion of the horizontal stripes); (7)cleavage site (gray fill, diamond pattern and the left portion of thehorizontal stripes); (8) preproelastase protein (entire scheme); (9)proelastase protein (diagonal stripes, gray fill, diamond pattern andhorizontal stripes combined); and (10) mature elastase protein(horizontal stripes). The table shows the amino acid designations of theregion in the schematic spanned by the arrow. Not drawn to scale. Thenomenclature is for reference purposes only and is not intended toconnote a particular function, activity or mechanism.

FIG. 3. Diagram of the pPROT24-V Vector. “α-factor secretion” refers toa cassette containing the yeast α-factor signal peptide and propeptide,followed by a Kex2 site and STE13 repeats.

FIG. 4: SDS-PAGE analysis of fractions from capture chromatography of a201-24-266-VU culture containing trypsin-activated pro-PRT-201. Lanenumbers correspond to fraction numbers. Fractions 6-18 primarily consistof glycosylated proenzyme (upper band) and non-glycosylated proenzyme(lower band). Fractions 19-35 primarily consist of non-glycosylatedproenzyme. M=molecular weight markers. FT=column flow through.

FIGS. 5A-5F: FIG. 5A shows an auto-activated proprotein data table.Propeptide sequences are listed in the first column. SDS-PAGE ofsupernatants after 1, 2 and 3 days of induction (lanes 1, 2 and 3,respectively) are shown in the second column. Relative proprotein yieldsbased on SDS-PAGE are listed in the third column. Relative stabilitiesof the proprotein over 3 days of induction based on SDS-PAGE are listedin the fourth column. Proproteins with 42 and 48 propeptide sequencesare ranked as having low stability because of the presence of matureprotein during induction (observed after 1, 2 and 3 days for the 42variant and after 2 and 3 days for the 48 variant). Relative conversionrates of the proproteins as determined by time to achieve maximal SLAPreaction velocity are listed in the fifth column. The estimatedpercentages of converted protein that comprised N-terminal variants ofthe mature elastase protein are listed in the sixth column. FIGS. 5B-5Fshow conversion rate data for propeptide sequences 24, 42, 48, 49 and55, respectively.

FIG. 6. pPROT55M3-V cloning scheme. pPROT55M3-V was engineered by invitro ligation of two additional expression cassettes to the 4.3 kbpPROT55-V vector backbone, giving a total of three tandem expressioncassettes. The 2.3 kb expression cassette fragment was released frompPROT55-V with a BglII and BamHI restriction digest and purified,followed by ligation of two copies of the expression cassette topPROT55-V linearized with BamHI.

FIGS. 7A-B: Shaker flask optimization of clone 201-55M3-006-VU. Thestandard induction media, BKME, was prepared and supplemented withsodium citrate to achieve final concentrations of 0, 12.5, 25 and 50 mMsodium citrate, pH 5.5. The media was used to resuspend growth phasecell pellets using a ratio of 1 g wet cell weight to 10 mL of inductionmedia. Cell suspensions of 25 mL each were placed in a 250 mLnon-baffled flasks and incubated at 22° C. or 25° C. for 3 days withshaking at 275 rpm. Methanol was added twice daily to a finalconcentration of 0.5% by volume. Supernatant aliquots were taken duringthe 3-day period and analyzed for protein expression by SDS-PAGE andCoomassie staining. FIG. 7A shows samples induced at 22° C. and FIG. 7Bshows samples induced at 25° C. In both FIG. 7A and FIG. 7B, lanes 1-3are supernatants after 1, 2 and 3 days of induction, respectively,containing 0% sodium citrate; lanes 4-6 are similar except with 12.5%sodium citrate; lanes 7-9 are similar except with 25% sodium citrate;and lanes 10-12 are similar except with 50 mM sodium citrate.

FIG. 8. SDS-PAGE analysis of 201-55-001-VU and 201-55M3-003-VUfermentation supernatants. Lanes 1, 2: 201-55-001-VU supernatant; lanes3, 4: 201-55M3-003-VU supernatant; lane 5: empty; lane 6: molecularweight markers.

FIG. 9. SDS-PAGE analysis of fractions from pPROT55M3-V proproteincapture chromatography. A total of 30 microliters from each elutionfraction was mixed with 10 microliters 4× Laemmli sample loading buffersupplemented with beta-mercaptoethanol. The proteins wereelectrophoresed on an 8-16% linear gradient gel followed by Coomassiestaining. Two predominant forms of PRT-201 were observed: the proprotein(fractions 15-43) and spontaneously converted mature PRT-201 (fractions15-44). Lane numbers correspond to fraction numbers. M, molecular weightmarker. BC, before column (pre-load) sample.

FIG. 10: HIC-HPLC analysis of purified proprotein conversion. Purifiedpro-PRT-201-55M3-003-VU was subjected to conversion at 26° C. The graphshows relative amounts of mature (full-length) PRT-201 and N-terminalvariants produced during the conversion.

FIG. 11: HIC-HPLC analysis of proprotein conversion in fermentationsupernatant effected by tangential flow filtration. Clarified201-55M3-003-VU fermentation supernatant was subjected to tangentialflow filtration with 100 mM Tris, pH 8.0, using constant volumediafiltration at ambient temperature with regenerated cellulosemembranes. The graph shows relative amounts of proprotein, mature(full-length) PRT-201 and N-terminal variants present at various timepoints during the conversion.

FIG. 12. SDS-PAGE analysis of fractions from pPROT55M3-V conversioncapture chromatography. A total of 30 microliters from each elutionfraction was mixed with 10 microliters 4× Laemmli sample loading buffersupplemented with beta-mercaptoethanol. The proteins wereelectrophoresed on an 8-16% linear gradient gel followed by Coomassiestaining. Two predominant forms of PRT-201 were observed: theglycosylated mature form (fractions 35-70), and the non-glycosylatedmature form (fractions 75-160). Lane numbers correspond to fractionnumbers. M, molecular weight marker. BC, before column (pre-load)sample.

FIG. 13: Concentration dependence of pro conversion. Purifiedpro-PRT-201 from the 201-55M3-003-VU clone (pro-PRT-201-55M3-003-VU) wassubjected to conversion at concentrations of 0.2, 1.0, 1.6, and 1.8mg/mL. Conversion reactions were monitored by HIC-HPLC in real-timeuntil the proprotein was ≦1% of the total protein. The graph shows therelative amounts of mature (full-length) PRT-201 (lightly shaded bars)and N-terminal variants (darkly shaded bars) produced during theconversions.

FIG. 14. DNA sequence of synthetic (i.e., recombinant) porcinepancreatic elastase type 1 (SEQ ID NO:113). The recombinant sequencecontains a 750 base pair coding region. Sacll and Xbal restriction sitesas underlined were incorporated to facilitate cloning. Stop codons arehighlighted. The pro-peptide sequence is in bold-face type.

FIG. 15. Amino acid sequence of synthetic (i.e., recombinant) porcinetype I pancreatic elastase (SEQ ID NO:114). The pro-peptide region is inbold-face type while the trypsin cleavage site is highlighted. Aftercleavage of the 10 amino acid pro-peptide, the resulting mature enzymeis 240 amino acids.

FIG. 16. Cloning scheme of porcine type I pancreatic elastase into PV-1vector. After synthesis of the porcine type I pancreatic elastaseproprotein coding region, it was cloned in the Blue Heron pUC vector. Inaddition to amplifying the coding sequence of porcine type I pancreaticelastase, PCR was used to incorporate XhoI and SacII restriction sitesfor cloning into the PV-1 vector. The PCR product was digested,gel-purified and ligated with PV-1 vector digested with XhoI and SacII,thus resulting in a pPROT101-24-V expression vector encodingtrypsin-activated porcine type I pancreatic elastase proprotein.

FIG. 17. Expression analysis of auto-activated pPROT101-42-V andtrypsin-activated pPROT101-24-V clones during methanol induction bySDS-PAGE. Shaker flask supernatants after 1 day of induction wereanalyzed on an 8-16% gradient gel followed by staining with Coomassiestaining. Lanes 1-10 contain supernatants from ten different clonestransformed with pPROT101-42-V. Lanes 11-12 contain supernatants fromtwo different clones transformed with pPROT101-24-V. M, molecular weightmarkers.

FIG. 18. Expression analysis of auto-activated pPROT101-49-V andpPROT101-55L-V clones during methanol induction by SDS-PAGE. Shakerflask supernatants after 1 and 2 days of induction were analyzed on an8-16% gradient gel followed by Coomassie staining. Lanes 1-2 containsupernatants from a pPROT101-49-V clone after 1 and 2 days of induction,respectively. Lanes 3-4 contain supernatants from a pPROT101-55L-V cloneafter 1 and 2 days of induction, respectively. M, molecular weightmarkers.

FIG. 19. Time course activation of pPROT101-49-V and pPROT101-55L-Vproteins by small-scale conversion assay as determined by SLAP elastaseactivity. Error bars represent ±SD of the mean (n=4).

FIG. 20. SDS-PAGE analysis of pPROT101-49-V and pPROT101-55L-Vsupernatants before and after small-scale conversion assay. Prior toelectrophoresis, the samples were mixed with citric acid, the reducingagent TCEP, and LDS sample buffer (Invitrogen, CA). The samples wereheated at 70° C. for 10 minutes. Lanes 1-2: pPROT101-49-V pre- andpost-conversion assay supernatant, respectively; lanes 3-4:pPROT101-55L-V pre- and post-conversion assay supernatant, respectively.M, molecular weight markers.

FIG. 21. TrypZean standard curve.

FIG. 22. A side, partially sectioned view of one embodiment of themedical device described in Section 5.9.

FIG. 23. A view similar to FIG. 22 that illustrates the movement of theactuators of the medical device.

FIG. 24. An end section view in the plane of line 2-2 in FIG. 22.

FIG. 25. An end section view in the plane of line 3-3 in FIG. 22.

FIG. 26. A diagram of the fluid path of the medical device of FIG. 22,extending from the Luer hubs through the fluid delivery conduits to thereservoir and then to the tissue penetrators.

FIG. 27. A side, partially sectioned view of a second embodiment of themedical device of the present invention showing the actuators in theirconstrained configurations.

FIG. 28. A view similar to FIG. 27, but showing the actuators in theirunconstrained configurations.

FIG. 29. An end perspective view of the assembly along the line 3′-3′ ofFIG. 27.

FIG. 30. An end perspective view of the assembly along the line 4′-4′ ofFIG. 29 showing the tissue penetrators.

FIG. 31. A side view showing the detail of the proximal end of thedevice, shown to the right in FIGS. 27 and 28.

FIG. 32. A partial view of the exterior of the medical device of FIG. 22in its constrained position.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for recombinant expressionand production of mature, biologically active elastase proteins. Thepresent invention provides novel, efficient methods of makingrecombinant elastase proteins by culturing host cells, including thepreferred host cell, Pichia pastoris, comprising nucleic acids encodingproelastase proteins and preproelastase proteins. The use of therecombinant proteins to manufacture pharmaceutical compositions for thetreatment and prevention of diseases of biological conduits (includingarteries or veins) is also provided.

In certain aspects, the present invention is directed to recombinantauto-activated proelastase proteins and related nucleic acids, hostcells, and methods of manufacture. Such auto-activated proelastaseproteins are engineered to contain an elastase recognition siteimmediately N-terminal to the first residue of the mature elastaseprotein. Under specified culture conditions, such as those described inSection 6 below, it is possible to reduce auto-activation untilactivation is desired. It is also possible to reduce auto-activationuntil the pro-elastase is removed from the cell culture.

The present invention provides efficient expression and purificationprocesses for producing pharmaceutical grade elastase proteins. Thepresent invention also provides methods for treating or preventingdiseases of biological conduits using the elastase proteins of theinvention.

The description in Section 5 herein is applicable to the embodiments ofSection 8. Thus, for example, a reference to an elastase protein of theinvention includes, but is not limited to, a reference to an elastaseprotein according to any one of embodiments 1-39 and 68-69, or anelastase protein obtained or obtainable by the method of any one ofembodiments 89-224, 261 to 276, and 347 to 373. Likewise, a reference toa nucleic acid of the invention refers, inter alia, to a nucleic acidaccording to any one of embodiments 40-67; reference to a vector refers,inter alia, a reference to a vector according to any one of embodiments70-72; reference to a cell refers, inter alia, to a cell according toany one of embodiments 73-87, reference to a cell culture supernatantrefers, inter alia, to a cell culture supernatant according toembodiment 88; reference to compositions, such as pharmaceuticalcompositions, elastase formulations and unit dosages, includes, forexample, those exemplified in embodiments 277-314, 346, 386, and 413-420or those obtained or obtainable by the method of any one of embodiments261-276 and 374-385; and references to therapeutic methods also includesa reference to therapeutic methods according to any one of embodiments387-414; and reference to a kit includes a reference to, inter alia, areference to a kit of embodiments 421-425 of Section 8.

5.1 Elastase Proteins

The present invention is directed to, inter alia, methods forrecombinant expression and production of mature, biologically activeelastase proteins. The elastase proteins are generally expressed aspreproproteins, containing, among other sequences, a signal peptide, anactivation peptide, and a mature portion with biological activity.Suitable mature elastase protein sequences are described in Section5.1.1 below. Suitable activation peptide sequences are described inSection 5.1.2 below. Suitable signal sequences are described in Section5.1.3 below.

Accordingly, in certain aspects, the elastase proteins of the inventionare preproelastase proteins. Removal of the signal sequence from thepreproprotein upon secretion generally yields an inactive proproteincontaining an activation peptide and a mature protein. The phrases“activation sequence” and “activation peptide” are used interchangeablyherein. Thus, in other aspects, the elastase proteins of the inventionare proelastase proteins comprising an activation peptide that isoperably linked to a mature elastase protein. In an exemplaryembodiment, an activation peptide or sequence of a wild-type human typeI pancreatic elastase comprises the first 10 N-terminal amino acids ofthe human type I elastase proprotein (SEQ ID NO:22). In certainembodiments, the activation peptide is a peptide of SEQ ID NO:80 or SEQID NO:121. Activation peptides or sequences useful in the practice ofthis invention also include, but are not limited to, SEQ ID NO: 23, 72and 73. Still other activation sequences useful in the practice of thisinvention can be obtained from the N-terminal residues 1-10 of SEQ IDNO:64-69 and 98-103.

Removal of the activation peptide from the proelastase sequencegenerates a mature elastase protein. The step by which the activationpeptide is removed from the proelastase sequence/separated from themature elastase sequence to generate a mature elastase protein isreferred to herein as an activation step. Thus, in yet other aspects,the elastase proteins of the invention are mature elastase proteins.

Amino acid residues comprising the C-terminus (i.e., carboxy terminus)of the activation peptide and the N-terminus (i.e., amino terminus) ofthe mature protein that surround the cleavage bond are depicted in FIG.2 and also identified herein as follows. First, residues located at theC-terminus of the activation peptide are designated PX, . . . P5, P4,P3, P2, and P1, where P1 is the C-terminal residue of the activationpeptide. Residues located at the N-terminus of the mature protein aredesignated P1′, P2′, P3′, . . . PX′, where P1′ is the N-terminal aminoacid residue of the mature protein. The scissile bond that is cleaved byproteolysis (referred to as the “cleavage bond” in FIG. 2) is thepeptide bond between the P1 residue of the activation peptide and P1′residue of the mature protein.

In certain preferred embodiments, a proelastase protein of thedisclosure has a histidine residue at the P5 position and/or a prolineresidue at the P2 position and/or an alanine residue at the P1 position.

The region spanning 4 amino acids of the C-terminus of the activationpeptide (residues P4 to P1) through to the first 4 amino acids of theN-terminus of the mature protein (residues P1′ to P4′) is referred toherein as the “cleavage site.”

The region spanning approximately 5 amino acids of the C-terminus of theactivation peptide (i.e., residues P5 to P1) through to approximatelythe first 3 amino acids of the N-terminus of the mature protein (i.e.,residues P1′ to P3′) is referred to herein as the “cleavage domain.”Examples of cleavage domains that can be used in the context of thisinvention include, but are not limited to, SEQ ID NOS: 42, 43, 48, 49,52, 53, 54 or 55. Other cleavage domains that can be used in thisinvention are any of the sequences described by the consensus cleavagedomain sequence (SEQ ID NO:123) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈,where Xaa₁=P5, Xaa₂=P4, Xaa₃=P3, Xaa₄=P2, Xaa₅=P1, Xaa₆=P1′, Xaa₇=P2′,and Xaa₈=P3′, where:

-   -   Xaa₁ is glutamate, histidine, proline, glycine, asparagine,        lysine, or alanine, or, optionally, an analog thereof;    -   Xaa₂ is threonine, alanine, proline or histidine or, optionally,        an analog thereof;    -   Xaa₃ is alanine, leucine, isoleucine, methionine, lysine,        asparagine or valine, or, optionally, an analog thereof, but is        preferably not glycine or proline;    -   Xaa₄ is proline, alanine, leucine, isoleucine, glycine, valine,        or threonine, or, optionally, an analog thereof;    -   Xaa₅ is alanine, leucine, valine, isoleucine, or serine but not        glycine, tyrosine, phenylalanine, proline, arginine, glutamate,        or lysine, or, optionally, an analog thereof;    -   Xaa₆ is alanine, leucine, valine, isoleucine or serine, or,        optionally, an analog thereof;    -   Xaa₇ is glycine, alanine, or valine, or, optionally, an analog        thereof; and    -   Xaa₈ is glycine, valine, threonine, phenylalanine, tyrosine, or        tryptophan, or, optionally, an analog thereof.

In specific embodiments, the proelastase cleavage domain is any sequencedescribed by the consensus cleavage domain sequence of SEQ ID NO:74, SEQID NO:123, or SEQ ID NO:125.

In certain aspects, the proelastase proteins have a propeptide portionof the cleavage domain of are any of the sequences described by theconsensus cleavage domain sequence (SEQ ID NO:126) Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅, where Xaa₁=P5, Xaa₂=P4, Xaa₃=P3, Xaa₄=P2, Xaa₅=P1, where:

-   -   Xaa₁ is any natural amino acid;    -   Xaa₂ is any natural amino acid except glycine, lysine,        phenylalanine, tyrosine, tryptophan, or arginine;    -   Xaa₃ is any natural amino acid;    -   Xaa₄ is proline, alanine, leucine, isoleucine, glycine, valine,        histidine, asparagine, or threonine, and    -   Xaa₅ is alanine, leucine, valine, isoleucine, asparagine, or        serine.

In certain aspects, Xaa₁ is glutamate or histidine, preferably histidineand/or Xaa₄ is proline and/or Xaa₅ is alanine.

The three amino acid region spanning residues P3, P2, and P1 of theactivation peptide is referred to herein as an “elastase recognitionsite”. Examples of recognition sites that can be used in the context ofthis invention include, but are not limited to, SEQ ID NOS: 14-16, and18-21. Other recognition sites contemplated by this invention includeany recognition site described by the consensus recognition sites of SEQID NO: 11, 12, 13, or 93. The SEQ ID NO:11 consensus elastaserecognition sequence 1 is represented by the peptide sequence Xaa₁ Xaa₂Xaa₃, wherein Xaa₁=P3, Xaa₂=P2, Xaa₃=P1, wherein:

-   -   Xaa₁ is alanine, leucine, isoleucine, methionine, lysine,        asparagine or valine, or, optionally, an analog thereof but is        preferably not glycine or proline;    -   Xaa₂ is proline, alanine, leucine, isoleucine, glycine, valine,        or threonine, or, optionally, an analog thereof;    -   Xaa₃ is alanine, leucine, valine, isoleucine, or serine, or,        optionally, an analog thereof, but is preferably not glycine,        tyrosine, phenylalanine, proline, arginine, glutamate, or        lysine.

The SEQ ID NO:12 consensus elastase recognition sequence 2 isrepresented by the sequence Xaa₁ Pro Xaa₂, wherein:

-   -   Xaa₁ is alanine, leucine, isoleucine, methionine, lysine, or        valine, or, optionally, an analog thereof, but is preferably not        glycine or proline;    -   Pro is proline, or, optionally, an analog thereof;    -   Xaa₂ is alanine, leucine, valine, isoleucine, or serine, or,        optionally, an analog thereof, but is preferably not glycine,        tyrosine, phenylalanine, proline, arginine, glutamate, or        lysine.

The SEQ ID NO:13 consensus elastase recognition sequence 3 isrepresented by the peptide sequence Xaa₁ Xaa₂ Xaa₃, wherein Xaa₁=P3,Xaa₂=P2, Xaa₃=P1, wherein Xaa₁ is asparagine or alanine, or, optionally,an analog thereof; wherein Xaa₂ is proline or alanine, or, optionally,an analog thereof, and wherein Xaa₃ is alanine, leucine, or valine, or,optionally, an analog thereof.

The SEQ ID NO:93 consensus elastase recognition sequence 4 isrepresented by the sequence Xaa₁ Pro Xaa₂, wherein:

-   -   Xaa₁ is alanine, leucine, isoleucine, methionine, lysine,        asparagine or valine, or, optionally, an analog thereof, but is        preferably not glycine or proline;    -   Pro is proline, or, optionally, an analog thereof;    -   Xaa₂ is alanine, leucine, valine, isoleucine, or serine, or,        optionally, an analog thereof, but is preferably not glycine,        tyrosine, phenylalanine, proline, arginine, glutamate, or        lysine.

The SEQ ID NO:119 consensus elastase recognition sequence 5 isrepresented by the sequence Xaa₁ Xaa₂ Xaa₃, wherein:

-   -   Xaa₁ is any natural amino acid, but is preferably not glycine or        proline;    -   Xaa₂ is proline, alanine, leucine, isoleucine, glycine, valine,        histidine or threonine;    -   Xaa₃ is alanine, leucine, valine, isoleucine, or serine.

The SEQ ID NO:124 consensus elastase recognition sequence 5 isrepresented by the sequence Xaa₁ Xaa₂ Xaa₃, wherein:

-   -   Xaa₁ is alanine, leucine, isoleucine, methionine, lysine,        asparagine, histidine, or valine;    -   Xaa₂ is proline, alanine, leucine, isoleucine, glycine, valine,        or threonine;    -   Xaa₃ is alanine, leucine, valine, isoleucine, or serine.

In certain aspects, a proelastase protein of the disclosure has asequence for P10 through P3′ described by the consensus sequence of SEQID NO:122.

Reference to a sequence as a “cleavage sequence,” “cleavage domain,”“activation sequence,” “elastase recognition sequence,” etc., is solelyfor ease of reference and is not intended to imply any function of thesequence or mechanism by which the sequence is recognized or processed.

The proteins of the invention are generally composed of amino acids andmay in addition include one or more (e.g., up to 2, 3, 4, 5, 6, 7, 8, 9,10, 12 or 15) amino acid analogs. Generally, as used herein, an aminoacid refers to a naturally-occurring L stereoisomer. An amino acidanalog refers to a D-stereoisomer, a chemically modified amino acid, orother unnatural amino acid. For example, unnatural amino acids include,but are not limited to azetidinecarboxylic acid, 2-aminoadipic acid,3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid,4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. Achemically modified amino acid includes an amino acid that is chemicallyblocked, reversibly or irreversibly, and/or modified at one or more ofits side groups, α-carbon atoms, terminal amino group, or terminalcarboxylic acid group. A chemical modification includes adding chemicalmoieties, creating new bonds, and removing chemical moieties. Examplesof chemically modified amino acids include, for example, methioninesulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteinesulfone. Modifications at amino acid side groups include acylation oflysine ε-amino groups, N-alkylation of arginine, histidine, or lysine,alkylation of glutamic or aspartic carboxylic acid groups, anddeamidation of glutamine or asparagine. Modifications of the terminalamino include the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal carboxy group include theamide, lower alkyl amide, dialkyl amide, and lower alkyl estermodifications. A lower alkyl is a C₁-C₄ alkyl. Furthermore, one or moreside groups, or terminal groups, may be protected by protective groupsknown to the ordinarily-skilled protein chemist. The α-carbon of anamino acid may be mono- or di-methylated.

The proteins of the invention may be modified or derivatized, such asmodified by phosphorylation or glycosylation, or derivatized byconjugation, for example to a lipid or another protein (e.g., fortargeting or stabilization), or the like.

The present invention often relates to an “isolated” or “purified”elastase protein. An isolated elastase protein is one that is removedfrom its cellular milieu. A purified elastase protein is substantiallyfree of cellular material or other contaminating proteins from the cellor tissue source from which the elastase protein is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of elastase protein in which the proteinis separated from cellular components of the cells from which it isrecombinantly produced. Thus, elastase protein that is substantiallyfree of cellular material includes preparations of elastase proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the elastase protein is produced by a process in whichit is secreted into culture medium, it is also preferably substantiallyfree of the culture medium, i.e., culture medium represents less thanabout 20%, 10%, or 5% of the volume of the elastase protein preparation.

In certain embodiments, an isolated or purified elastase is additionallyfree or substantially free of cellular DNA. In specific embodiments,host cell genomic DNA is present in an amount of less than 10 picogram,less than 5 picograms, less than 3 picograms, less than 2 picograms, orless than 1 picogram of DNA per milligram of elastase protein in apreparation of isolated or purified elastase protein, or in acomposition comprising isolated or purified elastase protein. In oneembodiment, the host cell DNA is Pichia pastoris DNA.

Useful elastase protein sequences are provided in Table 1. In a specificembodiment, the invention provides a proelastase protein (including butnot limited to a protein of any one of SEQ ID NOS:6-9, 64-69, 88-91 and98-103) comprising (i) an activation sequence comprising an elastaserecognition sequence operably linked to (ii) the amino acid sequence ofa protein having type I elastase activity. Several polymorphisms ofhuman type I elastase are known. Any combination of polymorphisms iscontemplated in the proelastase protein sequences of the presentinvention, including but not limited to the combinations ofpolymorphisms set forth in Table 2. The protein optionally furthercomprises a signal sequence operably linked to said activation sequence.In certain specific embodiments, the signal sequence is operable inPichia pastoris, such as a yeast α-factor signal peptide, exemplified bythe amino acid sequence of SEQ ID NO:34. Alternative signal peptidecontaining sequences are exemplified in SEQ ID NOS:50 and 96 (containinga signal peptide, a non-elastase propeptide and a spacer sequence) andSEQ ID NOS:51 and 97 (containing the signal peptide and a non-elastasepropeptide). In other specific embodiments, the signal sequence is amammalian secretion signal sequence, such as a porcine elastase signalsequence. Preferably, the elastase recognition sequence is a type Ielastase recognition sequence, most preferably a human type I elastaserecognition sequence.

The present invention further encompasses variants of the elastaseproteins of the invention. Variants may contain amino acid substitutionsat one or more predicted non-essential amino acid residues. Preferably,a variant includes no more than 15, no more than 12, no more than 10, nomore than 9, no more than 8, no more than 7, no more than 6, no morethan 5, no more than 4, no more than 3, no more than 2 or no more than 1conservative amino acid substitution relative to a naturally occurringmature elastase and/or no more than 5, no more than 4, no more than 3,or no more than 2 non-conservative amino acid substitutions, or no morethan 1 non-conservative amino acid substitution, relative to a naturallyoccurring mature elastase.

In a specific embodiments, the variant has no more than 10 or morepreferably no more than five conservative amino acid substitutionsrelative to a mature elastase, a proelastase or a preproelastase of theinvention, such as with respect to a mature elastase of SEQ ID NO:1 orSEQ ID NO:84 or a proelastase protein of SEQ ID NO:6-9, 64-69, 88-91,and 98-103. The amino acid sequences of SEQ ID NOS:1 and 84 contain oneor more positions corresponding to potential polymorphisms in the matureelastase sequence, at positions 44 (W or R); 59 (M or V); 220 (V or L);and 243 (Q or R) (positions refer to preproprotein). The invention thusencompasses mature elastase proteins with any combination of the fourpolymorphisms identified in SEQ ID NO:84. Each of such combinations isoutlined in Table 3 above. The sequence of SEQ ID NOS:88-91 and 98-103further contain a potential polymorphism in the propeptide sequence, atposition 10 (Q or H). The invention thus encompasses preproelastase andproelastase sequences containing any combination of the fivepolymorphisms identified in SEQ ID NOS:88-91 and 98-103. Each of suchcombinations is outlined in Table 2 above.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan).

The variant elastase proteins of the invention may include amino acidsubstitutions with amino acid analogs as well as amino acids, asdescribed herein.

In specific embodiments, the protein of the invention comprises orconsists essentially of a variant of a mature human type I elastase,e.g., a variant which is at least about 75%, 85%, 90%, 93%, 95%, 96%,97%, 98% or 99% identical to the elastase proproteins or mature elastaseproteins listed in Table 1, such as, but not limited to, the elastaseproproteins of SEQ ID NOS: 6-9, 64-69, 88-91, and 98-103, and retainelastase activity when expressed to produce a mature elastase protein ofSEQ ID NO: 1, 4, 5, 84 or 87.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (% identity=(# ofidentical positions/total # of overlapping positions)×100). In oneembodiment, the two sequences are the same length. In other embodiments,the two sequences differ in length by no more than 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9% or 10% of the length of the longer of the two sequences.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and) (BLAST programs of Altschul, et al.(1990) J Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Seewww.ncbi.nlm.nih.gov.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the CGC sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art and include ADVANCE and ADAM asdescribed in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5;and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. For a further description of FASTA parameters, seebioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2, the contents of whichare incorporated herein by reference.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

The elastase proteins of the invention can exhibit post-translationalmodifications, including, but not limited to glycosylations (e.g.,N-linked or O-linked glycosylations), myristylations, palmitylations,acetylations and phosphorylations (e.g., serine/threonine or tyrosine).In one embodiment, the elastase proteins of the invention exhibitreduced levels of 0-linked glycosylation and/or N-linked glycosylationrelative to endogenously expressed elastase proteins. In anotherembodiment, the elastase proteins of the invention do not exhibitO-linked glycosylation or N-linked glycosylation.

5.1.1. The Mature Elastase Sequence

The mature elastase sequences of the present invention are preferablymammalian elastase sequences, most preferably human elastase sequences.In other embodiments, the mature mammalian elastase sequences are fromother mammals such as mouse, rat, pig, cow, or horse.

In the methods and compositions of the invention, the mature elastasesequence employed is preferably that of a type I pancreatic elastase,which preferentially cleaves hydrophobic protein sequences, preferableon the carboxy side of small hydrophobic residues such as alanine.Examples of type I pancreatic elastases include the human elastase Ienzyme (NCBI Accession Number NP_001962) that is expressed in skin andthe porcine pancreatic elastase I enzyme (NCBI Accession NumberCAA27670) that is expressed in the pancreas. SEQ ID NO:1 and SEQ IDNO:84 are examples of mature human type I elastase sequences.

Alternatively, a type II elastase that can cleave hydrophobic proteinsequences, preferably on the carboxy side of medium to large hydrophobicamino acid residues, may be used. Examples of type II elastases includethe human elastase IIA enzyme (NCBI Accession Number NP254275) and theporcine elastase II enzyme (NCBI Accession Number A26823) that are bothexpressed in the pancreas.

Variants of a mature elastase protein of the invention are alsoencompassed. Variants include proteins comprising amino acid sequencessufficiently identical to or derived from the amino acid sequence of themature elastase protein of the invention and exhibit elastase biologicalactivity. A biologically active portion of a mature elastase protein ofthe invention can be a protein which is, for example, at least 150, 160,175, 180, 185, 190, 200, 210, 220, 230, 231, 232, 233, 234, 235, 236,237, 238, or 239 amino acids in length. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe functional activities of the native form of a mature elastaseprotein of the invention.

In addition, mature elastase proteins comprising any combination of thefour human type I elastase polymorphisms are represented by SEQ IDNO:84. Possible combinations are set forth in Table 3 above.

5.1.2. Proelastase Activation Sequences

The elastase activation sequence is any sequence whose removal from aproelastase protein results in a biologically active mature elastaseprotein.

Activation sequences generally contain protease recognition sitesadjacent to where proproteins are cleaved to produce mature,biologically active proteins. An activation sequence may be engineeredto add a protease or elastase recognition site, or it may be engineeredto replace an existing protease recognition site with another proteaserecognition site. Activation peptides or sequences useful in thepractice of this invention include, but are not limited to, SEQ ID NO:23, 72 and 73. Still other activation sequences useful in the practiceof this invention can be obtained from the N-terminal residues 1-10 ofSEQ ID NO:64-68. In preferred aspects, the proelastase activationsequence is engineered to contain a recognition sequence for a type I ortype II elastase. Most preferably, the elastase recognition sequence isrecognized by the mature elastase to which it is operably linked. Thus,in embodiments directed to a type II elastase, the recognition sequenceis most preferably a type II elastase recognition sequence. Conversely,in embodiments directed to a type I elastase, the recognition sequenceis most preferably a type I elastase recognition sequence. In apreferred embodiment, the recognition sequence is a human type Ielastase recognition sequence. Exemplary type I recognition sequencesinclude the amino acid sequence of SEQ ID NOS: 14-16, and 18-21. Otherrecognition sites contemplated by this invention include any recognitionsite described by the consensus recognition sites of SEQ ID NO: 11, 12,or 13.

5.1.3. Signal Sequences

The proelastase proteins of the invention may further contain a signalsequence which increases the secretion of a proelastase protein into theculture medium of the host cell in which it is expressed.

The native signal sequence of the elastase protein may be used,particularly for expression in a mammalian host cell. In otherembodiments, the native signal sequence of an elastase protein of theinvention can be removed and replaced with a signal sequence fromanother protein, such as the porcine type I elastase signal sequence,the human type I elastase signal sequence, or the yeast α-factor signalsequence. In certain specific embodiments, the yeast α-factor signalpeptide can further comprise (1) a yeast α-factor propeptide or (2) ayeast α-factor propeptide and spacer sequence, each respectivelyexemplified by the amino acid sequence of SEQ ID NOS:50 and 96 or SEQ IDNOS:51 and 97. Alternatively, the gp67 secretory sequence of thebaculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology (Ausubel et al., eds.,John Wiley & Sons, 1992)). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Sambrook et al., supra) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

5.2 Elastase Nucleic Acids

One aspect of the invention pertains to recombinant nucleic acidmolecules that encode a recombinant elastase protein of the invention.As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

The present invention is directed to nucleic acids encoding the elastaseproteins of the invention. Thus, in certain embodiments, the presentinvention provides a nucleic acid molecule comprising a nucleotidesequence which encodes a proelastase protein (including but not limitedto a protein of any one of SEQ ID NOS:6-9, 64-69, 88-91, or 98-103)comprising (i) an activation sequence comprising an elastase recognitionsequence operably linked to (ii) the amino acid sequence of a proteinhaving type I elastase activity. In other embodiments, the presentinvention also provides a nucleic acid molecule comprising a nucleotidesequence which encodes a protein comprising (i) a signal sequenceoperable in Pichia pastoris operably linked to (ii) an activationsequence (including but not limited to an amino acid sequence of SEQ IDNOS: 23, 72, or 73) comprising a protease recognition sequence which inturn is operably linked to (iii) the amino acid sequence of a maturehuman type I elastase.

A nucleic acid of the invention may be purified. A “purified” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

In instances wherein the nucleic acid molecule is a cDNA or RNA, e.g.,mRNA, molecule, such molecules can include a poly A “tail,” or,alternatively, can lack such a 3′ tail.

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

5.3 Recombinant Expression Vectors and Host Cells

Further provided are vectors comprising any of the nucleic acids of theinvention or host cells engineered to express the nucleic acids of theinvention. In specific embodiments, the vectors comprise a nucleotidesequence which regulates the expression of the protein encoded by thenucleic acid of the invention. For example, the nucleotide sequenceencoding the protein of the invention can be operably linked to amethanol-inducible promoter.

Host cells comprising the nucleic acids and vectors of the invention arealso provided. In certain embodiments, the vector or nucleic acid isintegrated into the host cell genome; in other embodiments, the vectoror nucleic acid is extrachromosomal. A preferred host cell is a Pichiapastoris cell.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which refers to a circulardouble-stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of coding sequences towhich they are operably linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors).

The recombinant expression vectors of the invention comprise nucleotidesequence encoding a mature elastase, a proelastase or a preproelastaseof the invention in a form suitable for expression in a host cell. Thismeans that the recombinant expression vectors include one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operably linked to the nucleic acid sequence tobe expressed. Within a recombinant expression vector, “operably linked”is intended to mean that the nucleotide sequence of interest is linkedto the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185 (AcademicPress, San Diego, Calif., 1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cells and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of elastaseprotein desired, etc. The expression vectors of the invention can beintroduced into host cells to thereby produce elastase proteins encodedby nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of an elastase protein of the invention in prokaryotic (e.g.,E. coli) or eukaryotic cells (e.g., insect cells (using baculovirusexpression vectors), yeast cells or mammalian cells). Suitable hostcells are discussed further in Goeddel, supra. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression of therecombinant elastase protein; 2) to increase the solubility of therecombinant elastase protein; and 3) to aid in the purification of therecombinant elastase protein by acting as a ligand in affinitypurification. Often, in fusion expression vectors, a proteolyticcleavage domain is introduced at the junction of the fusion moiety andthe recombinant protein to enable separation of the recombinant proteinfrom the fusion moiety subsequent to purification of the fusion protein.Thus, the fusion moiety and proteolytic cleavage domain together can actas an activation sequence, including a protease recognition site, forrecombinant expression of an elastase protein. Enzymes capable ofactivating such fusion proteins, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc.; Smith andJohnson, 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET-11d (Studieret al., 1990, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif., 185:60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET-11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ, prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant elastase protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,1990, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. 185:119-129). Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., 1992, NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiae or P.pastoris include pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz etal., 1987, Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and pPicZ (Invitrogen Corp, San Diego, Calif.). For expressionin yeast, a methanol-inducible promoter is preferably used. Alterationof the nucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in P. pastoris is also contemplatedherein. More specifically, the codons of SEQ ID NO:33 or SEQ ID NO:81can be substituted for codons that are preferentially utilized in P.pastoris.

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39). Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in insect cells.

In yet another embodiment, an elastase protein is expressed in mammaliancells using a mammalian expression vector. Examples of mammalianexpression vectors include pCDM8 (Seed, 1987, Nature 329(6142):840-2)and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985,Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. EP264166). Developmentally-regulated promoters are alsoencompassed, for example the mouse hox promoters (Kessel and Gruss,1990, Science 249:374-379) and the beta-fetoprotein promoter (Campes andTilghman, 1989, Genes Dev. 3:537-546).

In certain aspects of the invention, expression of a protein of theinvention may be increased by increasing the dosage of the correspondinggene, for example by the use of a high copy expression vector or geneamplification. Gene amplification can be achieved in dihydrofolatereductase− (“dhfr−”) deficient CHO cells by cotransfection of the geneof interest with the dhfr gene and exposure to selective medium withstepwise increasing concentrations of methotrexate. See, e.g., Ausubelet al., Current Protocols in Molecular Biology Unit 16.14, (John Wiley &Sons, New York, 1996). An alternative method for increasing gene copynumber is to multimerize an expression cassette (e.g., promoter withcoding sequence) encoding the elastase protein of interest in a vectorprior to introducing the vector into the host cell. Methods and vectorsfor achieving expression cassette multimerization are known in yeast andmammalian host cell systems (see, e.g., Monaco, Methods in Biotechnology8: Animal Cell Biotechnology, at pp. 39-348 (Humana press, 1999);Vassileva et al., 2001, Protein Expression and Purification 21:71-80;Mansur et al., 2005, Biotechnology Letter 27(5):339-45. In addition,kits for multi-copy gene expression are commercially available. Forexample, a multi-copy Pichia expression kit can be obtained fromInvitrogen (Carlsbad, Calif.). The multimerization of an expressioncassette is exemplified in Example 6, infra.

Accordingly, other aspects of the invention pertain to host cells intowhich a recombinant expression vector of the invention has beenintroduced. The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a coding sequence for aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the open reading frame ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin, zeocin and methotrexate.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker coding sequence will survive, while the other cellsdie).

In another embodiment, the expression characteristics of an endogenouselastase coding sequence within a cell, cell line or microorganism maybe modified by inserting a DNA regulatory element heterologous to theelastase coding sequence into the genome of a cell, stable cell line orcloned microorganism such that the inserted regulatory element isoperatively linked with the endogenous gene

A heterologous sequence, containing a regulatory element, may beinserted into a stable cell line or cloned microorganism, such that itis operatively linked with and activates expression of an endogenouselastase gene, using techniques, such as targeted homologousrecombination, which are well known to those of skill in the art, anddescribed e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publication No.WO 91/06667, published May 16, 1991. The heterologous sequence mayfurther include the signal peptides, cleavage sequences and/oractivation sequences of the present invention.

5.4 Methods of Manufacturing Mature Elastase Proteins

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce an elastase protein of theinvention. Accordingly, the invention further provides methods forproducing an elastase protein of the invention using the host cells ofthe invention. In one embodiment, the method comprises culturing thehost cell of invention (into which a recombinant expression vectorencoding an elastase protein of the invention has been introduced) in asuitable medium such that the elastase protein is produced. In anotherembodiment, the method further comprises isolating the elastase proteinfrom the medium or the host cell.

The present invention further provides methods for producing immatureelastase proteins of the invention comprising culturing a host cellengineered to express a nucleic acid of the invention under conditionsin which the proelastase protein is produced. In certain embodiments,the preproelastase protein is also produced. The present inventionfurther provides methods for producing mature elastase proteins of theinvention comprising culturing a host cell engineered to express anucleic acid of the invention under conditions in which a proelastaseprotein is produced and subjecting the proelastase protein to activationconditions such that the mature elastase protein is produced.

Preferred culture conditions for producing the immature and matureproteins of the invention, particularly for the host cell Pichiapastoris, include a period of growth at a low pH. In specificembodiments, the low pH is a pH of 2-6, a pH of 2-5, a pH of 3-6, a pHof 3-5, a pH of 4-6, a pH of 3-4 or any range whose upper and lowerlimits are selected from any of the foregoing values. At the end of theculture period, the pH of the culture can be raised, preferably to a pHof 7-11, most preferably to a pH of 8.

Where the expression of a proelastase protein of the invention is underthe control of a methanol-inducible promoter, conditions for producingan immature or mature elastase protein of the invention may alsocomprise a period of methanol induction.

The elastase production methods of the invention may further comprisethe step of recovering the protein expressed by the host cell. Incertain instances, the protein recovered is a proelastase, containingthe activation sequence. In other instances, the protein recovered is amature elastase lacking the activation sequence. Under certainconditions, both proelastase and mature elastase proteins are produced.In other instances, the preproelastase is produced.

Preferably, particularly where it is desired to circumventauto-activation of an auto-activated proelastase, culture conditions forproelastase expression comprise a period of growth in sodium citrate,sodium succinate, or sodium acetate. In specific embodiments, aconcentration of about 5-50 mM, 7.5-100 Mm, 10-150 mM, 50-200 mM, 75-175mM, 100-150 mM, 75-125 mM, or of any range whose upper and lower limitsare selected from any of the foregoing values is used. In a preferredembodiment, the sodium citrate, sodium succinate, or sodium acetateconcentration is 90-110 mM, most preferably 100 mM.

Additionally, particularly where it is desired to circumvent proteindegradation, culture conditions for proelastase expression comprise aperiod of growth and induction at the lower end of the temperature rangesuitable for the host cell in question. For example, where the host cellis a Pichia pastoris host cell, the preferred range is about 22-28° C.In specific embodiments, the Pichia pastoris host cell is cultured at atemperature of about 28° C. The growth and induction need not beperformed at the same temperature; for example, in an embodiment wherePichia pastoris is utilized as a host cell, growth can be performed at28° C. while induction can be performed at 22° C.

The activation of an auto-activated proelastase protein of the inventionmay be initiated by the addition of extrinsic elastase in a small(catalytic) amount. Alternatively or concurrently, the activation of anauto-activated proelastase protein of the invention may be initiated byraising the pH of the solution containing the auto-activated proelastaseprotein. The pH is preferably 7-11; in specific embodiments, thesolution is at a pH of 7-10, 7-9, 8-10, 8-9, or any range whose upperand lower limits are selected from any of the foregoing values. In apreferred embodiment, the pH of the solution 7-9, most preferably 8.

In specific embodiments, the auto-activated proelastase may be subjectedto Tris base, during the activation step. In specific embodiments, Trisbase is added to a concentration of 5-50 mM, 7.5-100 Mm, 10-150 mM,50-200 mM, 75-175 mM, 100-150 mM, 75-125 mM, or of any range whose upperand lower limits are selected from any of the foregoing values. In apreferred embodiment, Tris base is added to a concentration 90-110 mM,most preferably 100 mM. The pH of the Tris base is preferably 7-11; inspecific embodiments, the Tris base is at a pH of 7-10, 7-9, 8-10, 8-9,or any range whose upper and lower limits are selected from any of theforegoing values. In a preferred embodiment, the Tris base is at a pH of7-9, most preferably 8.

In certain aspects of the invention, the temperature for elastaseautoactivation is ambient temperature, e.g., a temperature ranging from22° C. to 26° C. In certain embodiments, the elastase activation step ispreferably performed with the proelastase at a low initialconcentration, e.g., 0.1-0.3 mg/ml, for optimal accuracy of the cleavagereaction and minimal formation of N-terminal variants.

In certain embodiments of the invention, addition of catalytic amountsof elastase is not required to convert the auto-activated proelastase tomature elastase, as the proelastase can undergo autoproteolysis. Incertain embodiments, the rate of autoproteolysis is concentrationindependent. Without seeking to be limited by theory, it is believedthat concentration independent autoproteolysis of certain auto-activatedproelastase proteins is mediated via an intramolecular process where theproelastase molecule cleaves itself via an intramolecular reaction.However, in other embodiments, activation of the auto-activatedproelastase is concentration dependent. Without seeking to be limited bytheory, it is believed that concentration dependent autoproteolysis ofcertain auto-activated proelastase proteins is mediated via anintermolecular reaction where proelastase is cleaved by anotherproelastase and/or by a mature elastase. In still other embodiments,certain auto-activated elastase proproteins display a combination ofconcentration dependent and concentration independent activation. Inthose instances where auto-activation is concentration dependent, theproprotein can be maintained in a more dilute form to reduce activationif desired. Activation of elastase proproteins comprising the elastasepropeptide cleavage domain of SEQ ID NO:55 that include but are notlimited to the proprotein of SEQ ID NO:69 can be controlled bymaintaining such proproteins in a dilute form. The binding cleft ofmature type I elastases is hydrophobic. Activation of elastaseproproteins can also be controlled by placing amino acids (such ashistidine) in the activation peptide that have side chains that arecharged at a lower pH but are not charged at higher pH. In this way,such pro-proteins can be expressed and purified in low pH solutionswhere activation is very slow and then activated quickly by raising thepH to a level where elastase activity is high and the side chain of theamino acid (such as histidine) is not charged.

It is also recognized that certain undesirable N-terminal sequencevariants of mature elastase can accumulate in the course of producingmature elastase proteins of this invention. More specifically,proelastase proteins containing the SEQ ID NO:42 elastase propeptidecleavage domain that include the proprotein of SEQ ID NO:6 can yieldN-terminal sequence variants where cleavage has occurred at the peptidebond that is C-terminal to any of the residues at positions P3 or P2.However, the occurrence of such undesirable N-terminal variants can bereduced by placing certain amino acids in certain locations in theactivation sequence. For example, when a proline in present in the P2position, the production of N-terminal variants with one or twoadditional N-terminal amino acids is reduced or eliminated. Also,elimination of the need for trypsin for activation reduces or eliminatesthe production of the variant that lacks nine N-terminal residues.Additionally, the occurrence of undesirable N-terminal variants can bereduced or eliminated by conducting the activation reaction undercertain conditions.

More specifically, in certain embodiments activation conditions includean “extended conversion” step during which N-terminal variants producedduring the initial portion of the conversion reaction are subsequentlyselectively degraded. The relative amounts of protein species during“extended conversion” can be monitored in real-time by HIC-HPLC. Theselective degradation of undesired N-terminal variants increases theproportion of full-length, mature PRT-201 in the conversion reaction andreduces the proportion of N-terminal variants. For proelastase proteinscontaining the SEQ ID NO:55 elastase propeptide cleavage domain, theextended conversion step is performed for 4 to 8 hours, and preferablyabout 6 hr. For other proelastase proteins, the extended conversion stepmay be increased or decreased, depending on the proportion of N-terminalvariants relative to mature (full length) elastase immediately after allof the proelastase has been converted. When the conversion occurs incomplex media, such as fermentation broth, the period of extendedconversion may be increased due to competition at the active site ofmature elastase by other proteins and peptides in solution.Alternatively, a mixture of mature elastase and N-terminal variantelastase can be recovered from the complex media prior to the extendedconversion step, thereby reducing the active site competition and thetime required to remove the N-terminal variant species.

As mentioned above, during a conversion reaction forpro-PRT-201-55M3-003-VU, there is a side-reaction that leads to theproduction of N-terminal variants. In the specific case ofpro-PRT-201-55M3-003-VU, these N-terminal variants are missing the firsttwo valines and have little or no elastase activity. For other mutantpro-proteins there are additional N-terminal variants produced, somewith additions and others with different deletions. An N-terminalremoval step has been developed which reduces the N-terminal variants toa range of 0-2%. The development of this removal step evolved from avariety of experiments and observations. As mentioned previously, duringoptimization of conversion condition experiments with pro-PRT-201-42 itwas observed that longer conversion reactions often led to a very lowpercentage of N-terminal variants. It was subsequently determined thatthe longer conversion reactions allowed mature PRT-201 to selectivelydegrade N-terminal variants. The discovery of PRT-201's ability toselectively degrade N-terminal variants under certain conditions had atremendous benefit by helping to produce a more purified PRT-201 productwith less N-terminal variants. This N-terminal variant removal step wasimplemented into a large scale production process by establishingconditions that would allow the pro-PRT-201 to convert to mature PRT-201and then allow the PRT-201 to degrade the N-terminal variants.

A representative example of such a step is shown in FIG. 10 as it wasmonitored in real-time by HIC-HPLC. At approximately 50 minutes, the100% pro-PRT-201-55M3 had completely converted to approximately 86%mature PRT-201 and 14% N-terminal variants. The conversion reaction wasextended which allowed mature PRT-201 to selectively degrade theN-terminal variants resulting in a decrease of variants from 14% to 2%.With longer incubations, the N-terminal variants can be selectivelydegraded to an undetectable level. When the N-terminal variant level issufficiently low, the activity of PRT-201 is suppressed with sodiumcitrate and adjustment of the reaction pH to 5.0.

Once purified mature elastase has been obtained, the active enzyme canbe brought into a solution at which the elastase protein is relativelyinactive and placed into a buffer for further column chromatography,e.g., cation exchange chromatography, purification steps. In general,the elastase protein can be placed in sodium citrate at a concentrationof about 5 to 25 mM and a pH of about 2 to 5. In a specific embodiment,the elastase is placed in 20 mM sodium citrate, pH 5. The elutedfractions are then optionally analyzed by one, more than one, or all ofthe following methods: (1) spectrophotometry at A280 to determineconcentration, (2) SDS-PAGE to assess purity, (3) activity assay, e.g.,SLAP assay, to assess elastase specific activity, and (4) HIC-HPLC todetect mature elastase and N-terminal variants, and fractions withsuitable characteristics (e.g., acceptable specific activity, acceptablylow levels (preferably absence) of detectable glycoforms, and acceptablylow levels (preferably absence) of detectable N-terminal variants) arepooled.

Once purified mature elastase has been obtained, the active enzyme canbe brought into a suitable solution for lyophilization. In general, theelastase protein can be placed into a buffer of 1× phosphate bufferedsaline (“PBS”) (137 mM sodium chloride, 10 mM sodium phosphate, 2.7 mMpotassium phosphate pH 7.4) prior to lyophilization. In certainembodiments, the elastase protein can be placed into a buffer of 0.1×PBS(13.7 mM sodium chloride, 1.0 mM sodium phosphate, 0.27 mM potassiumphosphate pH 7.4) prior to lyophilization.

Expression of a proelastase sequence can in some instances yield amixture of proelastase and mature elastase proteins. Thus, the presentinvention provides a composition comprising both a proelastase proteinand a mature elastase protein.

5.5 Pharmaceutical Compositions

The mature elastase proteins of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the elastase protein andpharmaceutically inert ingredients, for example a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Also contemplated as pharmaceuticallyinert ingredients are conventional excipients, vehicles, fillers,binders, disintegrants, solvents, solubilizing agents, and coloringagents. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with a mature elastase protein, usethereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

Accordingly, certain aspects of the present invention relate topharmaceutical compositions. In specific embodiments, the presentinvention provides a composition comprising (i) a therapeuticallyeffective amount of a mature human type I elastase and (ii) apharmaceutically acceptable carrier. The mature human type I elastasethat can be used in the composition include but are not limited to theproteins of SEQ ID NO:1, 4, 5, 84, 87. The mature human type I elastasemay contain any of the combinations of polymorphisms set forth in Table3 above.

In other embodiments, the present invention provides a pharmaceuticalcomposition comprising (i) a therapeutically effective amount of maturehuman type I elastase (ii) a pharmaceutically acceptable carrier, whichpharmaceutical composition is free of trypsin, or fragments of trypsin.In other embodiments, the pharmaceutical composition is substantiallyfree of trypsin or fragments of trypsin. As used herein, the phrase“free of trypsin” refers to a composition in which trypsin is not usedin any portion of the production process. As used herein, the phrase“substantially free of trypsin” refers to a composition wherein trypsinis present at a final percent (i.e., weight trypsin/total compositionweight) of no more than about 0.0025% or more preferably, less thanabout 0.001% on a weight/weight basis. As used herein, the phrase “freeof” trypsin refers to a composition in which the variant isundetectable, e.g., by means of an enzymatic assay or ELISA.

In certain aspects, a composition of the invention has less trypsinactivity than the equivalent of 3 ng/ml of trypsin as measured by a BENZassay, preferably less trypsin activity than the equivalent of 2.5 ng/mlof trypsin as measured by a BENZ assay, and even more preferably lesstrypsin activity than the equivalent of 2 ng/ml of trypsin as measuredby a BENZ assay. In a specific embodiment, the present inventionprovides a composition comprising a therapeutically effective amount ofelastase protein in which the trypsin activity is the equivalent of lessthan 1.6 ng/ml of trypsin as measured by a BENZ assay. Examples ofelastase compositions with less trypsin activity than the equivalent of1.6 ng/ml of trypsin as measured by a BENZ assay are provided in Example8 below. In certain embodiments, the ng/ml trypsin activity can beassayed in a liquid human type I elastase composition or preparationcontaining 1 mg/ml human type I elastase protein. Thus, the trypsinactivities may also be described in terms of milligrams of elastaseprotein, for example, less than 3 ng trypsin activity/mg elastaseprotein, less than 1.56 ng trypsin activity/mg elastase protein, etc.

The present invention further provides pharmaceutical compositions thatare either free or substantially free of undesirable N-terminal variantsof mature elastase. Undesirable N-terminal variants include, but are notlimited to, variants produced by cleavage at the peptide bond that isC-terminal to any of the residues at the P5, P4, P3, P2, P′1, P′2, P′3,P′4, P′6, and/or P′9 positions. Certain undesirable N-terminal variantsare produced by trypsin activation; others are produced byautoactivation of proelastase sequences that do not contained optimizedactivation sequences.

In certain embodiments, the pharmaceutical composition is free orsubstantially free of one, more than one or all N-terminal variants ofmature elastase that include but are not limited to SEQ ID NOS: 2, 3,37, 38, 70, 71, 85, 86, 94, 95, 104, 105, 106, 107, 108. In certainembodiments, the present invention provides a pharmaceutical compositioncomprising (i) a therapeutically effective amount of mature human type Ielastase (ii) a pharmaceutically acceptable carrier, whichpharmaceutical composition is free or substantially free of any proteinwith SEQ ID NOS: 2, 3, 37, 38, 70, 71, 85, 86, 94, 95, 104, 105, 106,107, or 108. In other embodiments, the pharmaceutical composition issubstantially free of N-terminal variants of mature elastase thatinclude but are not limited to SEQ ID NOS: 2, 3, 37, 38, 70, 71, 85, 86,94, 95, 104, 105, 106, 107, or 108. As used herein, the phrase “free of”a particular variant refers to a composition in which the variant isundetectable, e.g., by means of cation exchange HPLC assay, hydrophobicinteraction HPLC assay, or mass spectrometry combined with liquidchromatography. As used herein, the phrase “substantially free” refersto a composition wherein the N-terminal variant is present at a finalpercent (i.e. weight N-terminal variant/total composition weight) of atleast less than about 0.5%. In certain preferred embodiments, thecomposition that is substantially free of N-terminal variant is acomposition where the concentration of N-terminal variant is less thanabout 0.1% or less than about 0.01% or, more preferably, even less thanabout 0.001% on a weight/weight basis. In certain aspects, the presenceof N-terminal variants is detected by means of cation exchange HPLCassay, hydrophobic interaction HPLC assay, or mass spectrometry combinedwith liquid chromatography.

In certain specific embodiments, a pharmaceutical composition that isfree of N-terminal variants of SEQ ID NO:70, 71, 104 and 105 is producedby activation of a proelastase which does not contain an arginine in theP1 position and/or an alanine in the P2 position.

In other embodiments, the present invention provides a pharmaceuticalcomposition comprising (i) a therapeutically effective amount of maturehuman type I elastase (ii) a pharmaceutically acceptable carrier, whichpharmaceutical composition is substantially free of bacterial proteinsand/or is substantially free of mammalian proteins other than saidmature human type I elastase. As used herein, the phrase “substantiallyfree of mammalian proteins” or “substantially free of bacterialproteins” refers to a composition wherein such proteins are present at afinal percent (i.e. weight mammalian proteins (other than elastase and,optionally, a carrier protein such as albumin) or bacterialproteins/total composition weight) of at least less than about 0.5%. Incertain preferred embodiments, the composition that is substantiallyfree of such proteins is a composition where the concentration of theundesirable protein is less than about 0.1% or less than about 0.01%,or, more preferably, even less than about 0.001% on a weight/weightbasis.

In certain aspects, a pharmaceutical composition that is “free ofmammalian proteins” (other than elastase) contains elastase that isproduced from a recombinant cell line that is not a mammalian cell andwhere no protein with a mammalian sequence or substantially a mammaliansequence is present in any portion of the production process. In certainaspects, a pharmaceutical composition that is “free of bacterialproteins” contains elastase that is produced from a recombinant cellline that is not a bacterial cell and where no protein with a bacterialsequence or substantially a bacterial sequence is present in any portionof the production process.

The mature human type I elastases (including variants) of the inventionare most preferably purified for use in pharmaceutical compositions. Inspecific embodiments, the elastases are at least 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% pure. In other specific embodiments, the elastasesare up to 98%, 98.5%, 99%, 99.2%, 99.5% or 99.8% pure.

For formulating into pharmaceutical compositions, the mature human typeI elastases of the invention preferably have a specific activity ofgreater than greater than 1, greater than 5, greater than 10, greaterthan 20, greater than 25, or greater than 30 U/mg of protein, asdetermined by measuring the rate of hydrolysis of the small peptidesubstrate N-succinyl-Ala-Ala-Ala-pNitroanilide (SLAP), which iscatalyzed by the addition of elastase. One unit of activity is definedas the amount of elastase that catalyzes the hydrolysis of 1 micromoleof substrate per minute at 30° C. and specific activity is defined asactivity per mg of elastase protein (U/mg). Preferably, a pharmaceuticalcomposition comprises a mature human type I elastase which has aspecific activity within a range in which the lower limit is 1, 2, 3, 4,5, 7, 10, 15 or 20 U/mg protein and in which the upper limit is,independently, 5, 10, 15, 20, 25, 30, 35, 40 or 50 U/mg protein. Inexemplary embodiments, the specific activity is in the range of 1-40U/mg of protein, 1-5 U/mg protein, 2-10 U/mg protein, 4-15 U/mg protein,5-30 U/mg of protein, 10-20 U/mg of protein, 20-40 U/mg of protein, orany range whose upper and lower limits are selected from any of theforegoing values.

The pharmaceutical compositions of the invention are preferably stable.In specific embodiments, a pharmaceutical composition (for example apharmaceutical composition prepared by lyophilization above) maintainsat least 50%, more preferably at least 60%, and most preferably at least70% of its specific activity after a week, more preferably after amonth, yet more preferably after 3 months, and most preferably after 6months of storage at 4° C. In specific embodiments, the pharmaceuticalcomposition maintains at least 75%, at least 80%, at least 85%, at least90% or at least 95% of its specific activity after a week, morepreferably after a month, yet more preferably after 3 months, and mostpreferably after 6 months of storage at 4° C.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods foradministering elastases to treat or prevent diseases of biologicalconduits are described in WO 2001/21574; WO 2004/073504; and WO2006/036804. The most preferred route of administration is parenteral,for example direct administration to the vessel wall, including localadministration to the external adventitial surface of surgically exposedvessels and local administration to the vessel wall using a drugdelivery catheter. Solutions or suspensions used for parenteraladministration can include the following components: a sterile diluentsuch as water for injection, saline solution, phosphate buffered salinesolution, sugars such as sucrose or dextrans, fixed oils, polyethyleneglycols, glycerine, propylene glycol, polysorbate-80 (also known asTWEEN® 80), or other synthetic solvents; antibacterial agents such asmethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas phosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. The parenteral preparation can beenclosed in ampules, disposable syringes or single or multiple dosevials made of glass or plastic. The parenteral preparation can also beenclosed in a drug delivery catheter. In all cases, the composition mustbe sterile.

In specific embodiments, a pharmaceutical composition of the inventionis a liquid formulation comprising one or more of the followingexcipients: dextrose (e.g., 2-10% w/v); lactose (e.g., 2-10% w/v);mannitol (e.g., 2-10% w/v); sucrose (e.g., 2-10% w/v); trehalose (e.g.,2-10% w/v); ascorbic acid (e.g., 2-10 mM); calcium chloride (e.g., 4-20mM); dextran-70 (e.g., 2-10% w/v); poloxamer 188 (e.g., 0.2-1% w/v);polysorbate-80 (e.g., 0.001-5% w/v, more preferably 0.1-5%); glycerin(e.g., 0.2-5% w/v); arginine (e.g., 2-10% w/v); glycine (e.g., 2-10%w/v); dextran-44 (e.g., 2-10% w/v); and dextran-18 (e.g., 2-10% w/v). Incertain embodiments, the concentration, singly or in the aggregate, ofdextrose, lactose, mannitol, sucrose, trehalose, dextran-70, glycerin,arginine, glycine, dextran-44 or dextran-18 is within a range in whichthe lower limit is 2.5, 4, 5, or 7% w/v and in which the upper limit is,independently, 4, 5, 6, 8, or 10% w/v.

A liquid formulation can be made by adding water to a dry formulationcontaining a mature elastase protein, one or more buffer reagents and/orone or more excipients. The dry formulation can be made by lyophilizinga solution comprising mature elastase protein, one or more bufferreagents, and/or one or more excipients.

A liquid formulation can be made, for example, by reconstitutinglyophilized elastase proteins of the invention with sterilized water ora buffer solution. Examples of a buffer solution include sterilesolutions of saline or phosphate-buffered saline. In a specificembodiment, after reconstitution of a dry formulation comprising matureelastase protein to the desired protein concentration, the solutioncontains approximately 137 mM sodium chloride, 2.7 mM potassiumphosphate, 10 mM sodium phosphate (a phosphate buffered salineconcentration that is considered 1×) and the pH of the solution isapproximately 7.4. In certain aspects, the dry formulation comprisingmature elastase protein also contains sodium, chloride, and phosphateions in amounts such that only water is needed for reconstitution.

A liquid formulation can be also be made, for example, by reconstitutinglyophilized elastase proteins with a buffer solution containing one ormore excipients. Examples of excipients include polysorbate-80 anddextran. In a specific embodiment, after reconstitution of a dryformulation comprising mature elastase protein to the desired proteinconcentration, the resulting solution contains approximately 137 mMsodium chloride, 2.7 mM potassium phosphate, 10 mM sodium phosphate,0.01% polysorbate-80, and the pH of the solution is approximately 7.4.The one or more excipients can be mixed with the mature elastase proteinbefore lyophilization or after lyophilization but before reconstitution.Thus, in certain aspects, the dry formulation comprising mature elastaseprotein also contains excipients such as polysorbate-80 or dextran.

In certain aspects, the present invention provides a liquid formulationcomprising: 0.001-50 mg/ml of mature elastase protein in a solution of137 mM sodium chloride, 2.7 mM potassium phosphate, 10 mM sodiumphosphate and comprising 5-10%, more preferably 6-9%, of an excipientselected from dextrose, lactose, mannitol, sucrose, trehalose,dextran-70, glycerin, arginine, glycine, dextran-44 or dextran-18.

In a specific embodiment, the present invention provides a liquidformulation comprising: 0.001-50 mg/ml of mature elastase protein in asolution of 137 mM sodium chloride, 2.7 mM potassium phosphate, 10 mMsodium phosphate with a pH of 7.4.

In another specific embodiment, the present invention provides a liquidformulation comprising: 0.001-50 mg/ml of mature elastase protein in asolution of 137 mM sodium chloride, 2.7 mM potassium phosphate, 10 mMsodium phosphate and comprising 0.01% polysorbate-80, with a pH of 7.4.

In another specific embodiment, the present invention provides a liquidformulation comprising: 0.001-50 mg/ml of mature elastase protein in asolution of 137 mM sodium chloride, 2.7 mM potassium phosphate, 10 mMsodium phosphate and comprising 0.01% polysorbate-80 and 8% dextran-18,with a pH of 7.4.

In another specific embodiment, the present invention provides a liquidformulation comprising: 0.001-50 mg/ml of mature elastase protein in asolution of 137 mM sodium chloride, 2.7 mM potassium phosphate, 10 mMsodium phosphate and comprising 8% dextran-18, with a pH of 7.4.

The liquid formulations of the invention preferably contain a finalconcentration of mature elastase proteins within a range in which thelower limit is 0.1, 0.5, 1, 2.5, 5, 10, 15 or 20 mg/ml and in which theupper limit is, independently, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250,500, 1000, or 1500 mg/ml.

In certain aspects, the present invention provides a liquid formulationcomprising:

0.0001-500 mg/ml (more preferably 1-100 mg/ml, and yet more preferably0.5-20 mg/ml) of mature elastase protein in a solution of0.5×PBS-1.5×PBS (more preferably 1×PBS), the solution comprising 5-10%(more preferably 6-9%) of an excipient selected from dextrose, lactose,mannitol, sucrose, trehalose, dextran-70, glycerin, arginine, glycine,dextran-44 or dextran-18 and having a pH of 6.5-8.5. In a specificembodiment, the liquid formulation comprises 0.5 mg/ml mature elastaseprotein and 8% dextran-18 in 1×PBS, pH 7.4. In a specific embodiment,the liquid formulation comprises 5 mg/ml mature elastase protein and 8%dextran-18 in 1×PBS, pH 7.4.

A liquid formulation of the invention preferably has an osmolalitywithin a range in which the lower limit is 100, 125, 150, 175, 200, 250or 275 mOsm/kg and in which the upper limit is, independently, 500, 450,400, 350, 325, 300, 275 or 250 mOsm/kg. In specific embodiments, theosmolarity of a liquid formulation of the invention preferably has anosmolality of approximately 125 to 500 mOsm/kg, more preferably ofapproximately 275 to 325 mOsm/kg, for example as measured by thefreezing point depression method.

It is especially advantageous to formulate parenteral compositions, suchas compositions that can be made into the liquid formulations of theinvention, in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of mature elastase proteincalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier.

As defined herein, a therapeutically effective amount of mature elastaseprotein (i.e., an effective dosage) ranges from about 0.0033 mg-200 mg.For vessels with smaller diameter and thinner walls, such as those in aradiocephalic arteriovenous fistula, smaller doses (such as of 0.0033mg-2.0 mg) are preferable. For vessels with a larger diameter andthicker walls such as femoral arteries, larger doses (such as 2.05-100mg) are preferable.

In certain embodiments, the pharmaceutical compositions can be includedin a container, pack, dispenser, or catheter. In still otherembodiments, the pharmaceutical compositions can be included in acontainer, pack, dispenser, or catheter together with instructions foradministration. Instructions for administration can be included inprinted form either within or upon a container, pack, dispenser, orcatheter. Alternatively, instructions for administration can be includedeither within or upon a container, pack, dispenser, or catheter in theform of a reference to another printed or internet accessible documentthat provides the instructions.

In certain embodiments, the pharmaceutical compositions can be includedin a container, pack, or dispenser. In still other embodiments, thepharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. Instructionsfor administration can be included in printed form either within or upona container, pack, or dispenser. Alternatively, instructions foradministration can be included either within or upon a container, pack,or dispenser in the form of a reference to another printed or internetaccessible document that provides the instructions.

The invention includes methods for preparing pharmaceuticalcompositions. Once a mature elastase is produced according to theinvention, it can be lyophilized and stored until it is reconstitutedinto a pharmaceutical formulation suitable for administration. In anexemplary embodiment, the present invention provides a method ofisolating a lyophilized mature human type I elastase comprising: (a)culturing a host cell, such as a Pichia pastoris host cell, engineeredto express a nucleic acid molecule encoding a preproelastase openreading frame under conditions in which the open reading frame isexpressed, wherein said open reading frame comprises nucleotidesequences encoding, in a 5′ to 3′ direction (i) a signal peptideoperable in Pichia pastoris; (ii) an activation sequence comprising anelastase recognition sequence; and (iii) the sequence of a mature type Ielastase protein, thereby producing a proelastase protein; (b)subjecting the proelastase protein to autoactivation conditions, therebyproducing a mature type I elastase, wherein the autoactivationconditions include, for example: (i) changing the pH of a solutioncontaining the proelastase protein, e.g., to a pH of 6.5-11, preferably8-9; or (ii) purifying the proelastase protein, for example, by ionexchange chromatography, and subjecting the solution extended conversionto remove N-terminal variants, thereby producing mature human type Ielastase; (c) optionally, purifying the mature human type I elastase,e.g., ion exchange chromatography step for polish chromatography; and(d) lyophilizing the mature type I elastase, thereby isolating alyophilized mature human type I elastase. The mature type I elastase ispreferably a human type I elastase. In certain aspects, the lyophilizedmature type I elastase is preferably more than 95% pure; in specificembodiments, the lyophilized mature type I elastase is more than 98% ormore than 99% pure.

The mature elastase proteins of the invention can be formulated intopharmaceutical compositions. Thus, in an exemplary embodiments, thepresent invention provides a method of generating a pharmaceuticalcomposition comprising a mature human type I elastase, said methodcomprising (i) isolating a lyophilized mature human type I elastaseaccording to the methods described above (e.g., in Section 5.4); and(ii) reconstituting the lyophilized mature human type I elastase in apharmaceutically acceptable carrier.

5.6 Effective Dose

The present invention generally provides the benefit of parenteral,preferably local, administration of recombinant elastase proteins, aloneor in combination with other agents, for treating or preventing diseasein biological conduits.

In certain embodiments, as an alternative to parenteral administration,oral administration of agents for treating or preventing disease inbiological conduits may be used.

Toxicity and therapeutic efficacy of the elastase proteins utilized inthe practice of the methods of the invention can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Such information can be used to more accurately determine useful dosesin humans.

5.7 Methods of Administration

The invention relates to pharmaceutical compositions comprising novelelastase proteins and methods of use thereof for preventing or treatingdisease in biological conduits. Such pharmaceutical compositions can beformulated in a conventional manner as described in Section 5.5 above.

The elastase compositions of the present invention can be administeredto the desired segment of the biological conduit being treated by adevice known to one of skill in the art to be acceptable for delivery ofsolutions to the wall of an artery or vein, e.g., a syringe, a drugdelivery catheter, a drug delivery needle, an implanted drug deliverypolymer, such as a sheet or microsphere preparation, an implantablecatheter, or a polymer-coated vascular stent, preferably aself-expanding stent.

In certain embodiments, the administration to the desired segment may beguided by direct visualization, ultrasound, CT, fluoroscopic guidance,MRI or endoscopic guidance.

In certain aspects of the present invention, administration of anelastase to a biological conduit comprises applying a liquid formulationof elastase directly to the external adventitial surface of a surgicallyexposed artery or vein. In specific aspects of this invention, theadministration is performed with a syringe.

In certain aspects of the present invention, administration of anelastase to a biological conduit comprises localizing a deliveryapparatus in close proximity to the segment of the biological conduit tobe treated. In some embodiments, during delivery of the elastase proteinby a delivery apparatus, a portion of the delivery apparatus can beinserted into the wall of the biological conduit. In some embodiments,the lumen of the biological conduit can be pressurized while theelastase protein is delivered to the pressurized segment of thebiological conduit. In some embodiments, the lumen of the biologicalconduit is pressurized by mechanical action. In some embodiments, thelumen of the biological conduit is pressurized with a balloon catheter.In some embodiments, pressure is applied to the inner wall of thebiological conduit by a self-expanding member which is part of acatheter or device. In some embodiments, the elastase protein isadministered and the pressurizing is performed by the same device. Insome embodiments, the biological conduit is surgically exposed and theelastase protein is delivered into the lumen or is applied to theexternal surface of the biological conduit in vivo. In embodimentsinvolving luminal delivery, blood flow through the vessel may be stoppedwith a clamp or to allow the elastase to contact the vessel wall forlonger time periods and to prevent inhibition of the elastase by serum.In some embodiments, the biological conduit is surgically removed andthe elastase is delivered to the luminal surface and/or to the externalsurface of the conduit in vitro. The treated conduit may then, incertain embodiments, be returned to the body.

In other aspects of the present invention, administration of an elastaseto a biological conduit entails the use of a polymer formulation that isplaced as a stent within the vessel to be treated, a clamp or stripapplied to the external surface of the biological conduit, or a wrap onor around the vessel to be treated, or other device in, around or nearthe vessel to be treated.

In yet other aspects of the present invention, an elastase ispercutaneously injected into a tissue region for purpose of dilatingarteries and/or vein within that region, including collateral arteries.In other aspects, an elastase is percutaneously injected directly intothe wall of an artery or vein or into the surrounding tissues for thepurpose of dilating a specific segment of vessel. In embodiments aimedat treatment of heart vessels, an elastase protein can be eitherpercutaneously delivered to the pericardial space or directly applied tosurgically exposed coronary vessels.

Medical devices that can be used to administer the elastase proteins ofthe invention to blood vessels are described in Section 5.9 below.

5.8 Kits

The present invention provides kits for practicing the methods of thepresent invention. A “therapeutic” kit of the invention comprises in oneor more containers one or more of the agents described herein as usefulfor treating or preventing disease in biological conduits, optionallytogether with any agents that facilitate their delivery. An alternativekit of the invention, the “manufacturing” kit, comprises in one or morecontainers one or more of the agents described herein as useful formaking recombinant elastase proteins.

The therapeutic kit of the invention may optionally comprise additionalcomponents useful for performing the methods of the invention. By way ofexample, the therapeutic kit may comprise pharmaceutical carriers usefulfor formulating the agents of the invention. The therapeutic kit mayalso comprise a device or a component of a device for performing thetherapeutic methods of the invention, for example a syringe or needle.The inclusion of devices such as an intramural or perivascular injectioncatheters or intraluminal injection catheters in the therapeutic kits isalso contemplated. In certain embodiments, the agents of the inventioncan be provided in unit dose form. In addition or in the alternative,the kits of the invention may provide an instructional material whichdescribes performance of one or more methods of the invention, or anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. Instructional materials can be included inprinted form either within or upon one or more containers of the kit.Alternatively, instructional materials can be included either within orupon one or more containers of the kit in the form of a reference toanother printed or internet accessible document that provides theinstructional materials.

In specific embodiments, a kit of the invention comprises a medicaldevice as described in Section 5.9 below.

The manufacturing kit of the invention may optionally compriseadditional components useful for performing the methods of theinvention.

5.9 Medical Devices Useful for Administration of Elastase Proteins

Provided herein are medical devices that can be used to administer theelastase proteins of the present invention to a biological conduit, suchas an artery or vein. Such devices are described below and inprovisional application No. 61/025,084, filed Jan. 31, 2008, provisionalapplication No. 61/025,463, filed Feb. 1, 2008, and provisionalapplication No. 61/075,710, filed Jun. 25, 2008, each of which isincorporated by reference herein in its entirety. The elastase proteinsof the present invention can also be administered to biological conduitsvia conventional catheters.

In one embodiment, a medical device useful for administration ofelastase proteins has a central longitudinal axis, and comprises one ormore actuators, wherein the one or more actuators can exist in aconstrained configuration in which a length of said one or moreactuators is oriented substantially parallel to the longitudinal axis ofsaid medical device and an unconstrained configuration in which at leasta portion of the length of said one or more actuators is orientedsubstantially non-parallel to the device's central longitudinal axis.After the device is positioned at a target site adjacent to the wall ofa biological conduit, one or more actuators (and if desired, all of theactuators) may be released from a constrained configuration andpermitted to adopt an unconstrained configuration, thereby makingcontact with the wall of the biological conduit. The one or moreactuators may be of any shape, and in preferred embodiments, themovement of the one or more actuators from the constrained configurationto the unconstrained configuration occurs upon release of a constrainingforce by the device operator but without the input by the operator ofany deforming forces to the device or the target tissue.

In a first specific embodiment, shown in FIG. 22, the device is a fluiddelivery catheter 10 comprising one or more actuators that are formed asa pair of elongate splines 12, 14, the intermediate regions of which aremovable between a constrained configuration which is orientedsubstantially parallel to the central longitudinal axis 15 of thecatheter assembly and an unconstrained configuration in which at least aportion of the pair of splines is oriented substantially non-parallel tosaid central longitudinal axis (see the left L and right R portions ofthe spline lengths in FIG. 23). The one or more splines 12, 14 may beconstructed as elongate bands or wires that each have opposite proximaland distal ends. In a preferred embodiment, the splines have flat,opposing interior surfaces 24, 26, and flat opposite facing exteriorsurfaces 28, 30. In this embodiment, the splines 12, 14 can translatebetween constrained positions and unconstrained positions, as shownrespectively in FIGS. 22 and 23. In one embodiment, the pair of splinesis positioned back-to-back in their constrained configurations as shownin FIG. 22.

The catheter 10 further comprises one or more tissue penetrators 16, 18secured to one or more surfaces of the one or more splines 12, 14, acentral catheter component 20 having an elongate length, and an exteriorcatheter component 22 that can shield the tissue penetrator orpenetrators during catheter movement within the biological conduit.

The tissue penetrators 16, 18 may be constructed of any suitablematerial. Preferred examples of such materials include, but are notlimited to, nickel, aluminum, steel and alloys thereof. In a specificembodiment, the tissue penetrators are constructed of nitinol.

The central catheter component 20 and the exterior catheter component 22may be constructed of materials typically employed in constructingcatheters. Examples of such materials include, but are not limited to,silicone, polyurethane, nylon, Dacron, and PEBAX™.

The actuators are preferably constructed of a flexible, resilientmaterial. In a preferred embodiment, the flexible, resilient material iscapable of being constrained upon the application of a constrainingforce, e.g., when the actuators are in the constrained configuration,and adopts its original unconstrained shape when the constraining forceis removed, e.g., when the actuators are in the unconstrainedconfiguration. Any such flexible, resilient material can be used,including but not limited to surgical steel, aluminum, polypropylene,olefinic materials, polyurethane and other synthetic rubber or plasticmaterials. The one or more actuators are most preferably constructed ofa shape memory material. Examples of such shape memory materialsinclude, but are not limited to, copper-zinc-aluminum-nickel alloys,copper-aluminum-nickel alloys, and nickel-titanium (NiTi) alloys. In apreferred embodiment, the shape memory material is nitinol. In apreferred embodiment, when the pair of splines assumes the unconstrainedconfiguration, the shape memory properties of the material from whicheach spline is formed cause the splines, without the application of anyexternal deforming force, to bow radially away from each other in asingle plane as shown in FIG. 23.

One or more of the splines (and preferably each of the splines) has aflexible fluid delivery conduit 32, 34 that extends along the length ofthe spline, or within the spline, as shown in FIG. 24. As the splines12, 14 move from their straight, constrained configurations to theirbowed, unconstrained configurations, the fluid delivery conduits 32, 34also move from straight configurations to bowed configurations. In oneembodiment, the fluid delivery conduits 32, 34 are separate tubularconduits that are secured along the lengths of the pair of splines 12,14. In another embodiment, the fluid delivery conduits are conduitsformed into or within the material of the splines.

One or more of the splines (and preferably each of the splines 12,14) isalso formed with a zipper rail 36, 38 that extends along a length of thespline (FIG. 24). The zipper rails 36, 38 are formed of either the samematerial as the splines 12, 14, or a material that flexes with thesplines 12, 14.

One or more of the tissue penetrators 16, 18 is secured to the exteriorsurfaces 28, 30 of the pair of splines 12, 14 (FIG. 24). The tissuepenetrators 16, 18 are connected to and communicate with the fluiddelivery conduits 32, 34 that extend along the lengths of the splines12, 14. The tissue penetrators 16, 18 are positioned to projectsubstantially perpendicular from the exterior surfaces 28, 30 of thesplines 12, 14. The tissue penetrators 16, 18 have hollow interior boresthat communicate with the fluid delivery conduits 32, 34 of the splines.The distal ends of the tissue penetrators have fluid delivery ports thatcommunicate with the interior bores of the tissue penetrators.

The device permits delivery of fluids into or through one or moredistinct layers of a wall of a biological conduit, for example avascular wall. The vascular wall comprises numerous structures andlayers, including the endothelial layer and basement membrane layer(collectively the intimal layer), the internal elastic lamina, themedial layer, and the adventitial layer. These layers are arranged suchthat the endothelium is exposed to the lumen of the vessel and thebasement membrane, the internal elastic lamina, the media, and theadventitia are each successively layered over the endothelium, asdescribed in U.S. Pat. App. Publication No. 2006/0189941A1. With themedical devices of the present invention, the depth to which the tissuepenetrators 16, 18 can penetrate is determined by the length of eachtissue penetrator 16, 18. For example, if the target layer is theadventitial layer, tissue penetrators 16, 18 having a defined lengthsufficient for penetration to the depth of the adventitial layer upondeployment of the device are used. Likewise, if the target layer is themedial layer, tissue penetrators 16, 18 having a defined lengthsufficient for penetration to the depth of the medial layer upondeployment of the device are used.

In specific embodiments, the length of tissue penetrators 16, 18 mayrange from about 0.3 mm to about 5 mm for vascular applications, or upto about 20 mm or even 30 mm for applications involving other biologicalspaces or conduits, for example in colonic applications. Tissuepenetrators 16, 18 preferably have a diameter of about 0.2 mm (33 gauge)to about 3.4 mm (10 gauge), more preferably 0.2 mm to 1.3 mm (about 33to 21 gauge). The distal tips of the tissue penetrators may have astandard bevel, a short bevel, or a true short bevel. In an alternativeembodiment, the tissue penetrators attached to any one spline are not ofidentical lengths, but may be configured such that their distal endsalign so as to be equidistant from the wall of the biological conduitwhen the medical device is in the unconstrained position, e.g., duringuse.

The central catheter component 20 has an elongate length with oppositeproximal and distal ends, shown to the left and right respectively inFIG. 22. In one embodiment, the central catheter component 20 has acylindrical exterior surface that extends along its elongate length. Theproximal ends of the splines 12, 14 are attached e.g., soldered orglued, to the distal end of the central catheter component 20, while thedistal ends of the splines 12, 14 are attached, e.g., soldered or glued,to a catheter guide tip 40. The tip 40 has a smooth exterior surfacethat is designed to move easily in the biological conduit. A guide wirebore 48 extends through the length of the central catheter 20 and tip40. The guide wire bore is dimensioned to receive a guide wire insliding engagement through the bore.

A pair of fluid delivery lumens 44, 46 extends through the interior ofthe central catheter component 20 for the entire length of the cathetercomponent (FIG. 25). At the distal end of the central catheter component20 the pair of fluid delivery lumens 44, 46 communicates with the pairof fluid delivery conduits 32, 34 that extend along the lengths of thesplines 12, 14 to the tissue penetrators 16, 18. A guide wire bore 48also extends through the interior of the central catheter component 20from the proximal end to the distal end of the central cathetercomponent (FIG. 25). The proximal end of the central catheter component20 is provided with a pair of Luer hubs 50, 52 (FIG. 22). In oneembodiment, each Luer hub 50, 52 communicates with one of the fluiddelivery lumens 44, 46 extending through the length of the centralcatheter. Each Luer hub 50, 52 is designed to be connected with a fluiddelivery source to communicate a fluid through each Luer hub 50, 52,then through each fluid delivery lumen 44, 46 extending through thecentral catheter component 20, then through each fluid delivery conduit32, 34 extending along the lengths of the pair of splines 12, 14, andthen through the tissue penetrators 16, 18 secured to each of the pairof splines. In another embodiment, each Luer hub 50, 52 independentlycommunicates with both of the fluid delivery lumens 44, 46 extendingthrough the length of the central catheter component. In thisconfiguration, a first fluid can be delivered through a first Luer hubto both tissue penetrators 16, 18 and a second fluid can be deliveredthrough a second Luer hub to both tissue penetrators 16, 18. Delivery offluid to both tissue penetrators from each Luer hub can be achieved byan independent conduit extending from each Luer hub to a distal commonreservoir 61 as shown in FIG. 32. This reservoir communicates with bothtissue penetrators 16, 18. Alternatively, in another embodiment, themedical device of the instant invention comprises only a single Luer hubconnected to a single fluid delivery lumen extending through the centralcatheter, which then is attached to a distal common reservoir,permitting the delivery of a single fluid to both tissue penetrators 16,18.

The exterior catheter component 22 has a tubular configuration thatsurrounds the pair of splines 12, 14 and a majority of the centralcatheter 20 (FIG. 22). The catheter component 22 has an elongate lengththat extends between opposite proximal and distal ends of the cathetercomponent shown to the left and right, respectively in FIG. 22. Thecatheter component distal end is dimensioned to engage in a secureengagement with the guide tip 40, where the exterior surface of the tip40 merges with the exterior surface of the catheter component 22 whenthe catheter component distal end is engaged with the tip. The tubularconfiguration of the catheter component 22 is dimensioned so that aninterior surface of the catheter component 22 is spaced outwardly of theplurality of tissue penetrators 16, 18 on the pair of splines 12, 14 inthe constrained positions of the pair of splines. The proximal end ofthe central catheter 20 extends beyond the proximal end of the cathetercomponent 22 when the catheter component distal end engages with thecatheter guide tip 40.

A mechanical connection 54 is provided between the exterior cathetercomponent 22 proximal end and the central catheter component 20 proximalend that enables the exterior catheter component to be moved rearwardlyalong the lengths of the pair of splines 12, 14 and the central cathetercomponent 20 causing the exterior catheter component 22 distal end toseparate from the guide tip 40 and pass over the pair of splines 12, 14,and forwardly over the length of the central catheter component 20 andover the lengths of the pair of splines 12, 14 to engage the exteriorcatheter component 22 distal end with the tip 40 (FIG. 22). Themechanical connection 54 could be provided by a handle or button thatmanually slides the exterior catheter component 22 over the centralcatheter component 20. The connection 54 could also be provided by athumbwheel or trigger mechanism. In addition, the connection 54 could beprovided with an audible or tactile indicator (such as clicking) of theincremental movement of the exterior catheter component 22 relative tothe central catheter component 20.

In one embodiment, the exterior catheter component 22 is provided with asingle zipper track 56 that extends along the entire length of one sideof the exterior catheter component 22 on the interior surface of theexterior catheter component (FIG. 24). The zipper track 56 in theinterior of the exterior catheter component 22 engages in a slidingengagement with the zipper rails 36, 38 at one side of each of thesplines 12, 14. Advancing the exterior catheter component 22 forwardlyalong the lengths of the central catheter component 20 and the pair ofsplines 12, 14 toward the guide tip 40 of the catheter assembly causesthe zipper track 56 of the exterior catheter component to slide alongthe rails 36, 38 of the pair of splines 12, 14. This moves the pair ofsplines 12, 14 from their bowed, unconstrained configuration shown inFIG. 23 toward their back-to-back, constrained configuration shown inFIG. 22. The engagement of the spline rails 36, 38 in the zipper track56 of the exterior catheter component 22 holds the pair of splines 12,14 in their back-to-back relative positions shown in FIG. 22. With theexterior catheter component 22 pushed forward over the central cathetercomponent 20 and the pair of splines 12, 14 to where the distal end ofthe exterior catheter component 22 engages with the guide tip 40, thetissue penetrators 16, 18 are covered and the catheter assembly of thepresent invention can be safely moved forward or backward in abiological conduit. The exterior catheter component 22 covers the tissuepenetrators 16, 18 projecting from the pair of splines 12, 14 and theengagement of the exterior catheter component 22 with the distal guidetip 40 provides the catheter assembly with a smooth exterior surfacethat facilitates the insertion of the catheter assembly into and througha biological conduit such as a blood vessel. In another embodiment, theexterior catheter component 22 is provided with two zipper tracks at 180degrees from each other that extend along the entire length of theexterior catheter component 22 on the interior surface and the splineshave rails on both sides.

A guide wire 58 is used with the catheter assembly (FIG. 22). The guidewire 58 extends through the central catheter component guide wire bore48, along the splines 12, 14, and through the guide tip outlet 42. Incertain embodiments, the guide wire 58 has a solid core, e.g., stainlesssteel or superelastic nitinol. The guide wire may be constructed ofradiopaque material, either in its entirety or at its distal portions(e.g., the most distal 1 mm to 25 mm or the most distal 3 mm to 10 mm).The guide wire 58 may optionally be coated with a medically inertcoating such as TEFLON®.

In use of this device, the guide wire 58 is positioned in the biologicalconduit by methods well known in the art. The guide wire 58 extends fromthe biological conduit, through the guide wire outlet 42 in the tip 40of the assembly, through the exterior shielding catheter 22 past thetissue penetrators 16, 18, and through the guide wire bore 48 of thecentral catheter 20. In other embodiments, the catheter assembly is arapid-exchange catheter assembly, wherein the guide wire lumen ispresent in the distal end of the guide tip 40 of the catheter, but doesnot extend throughout the entire length of the medical device.

After positioning of the guide wire, the device is advanced into thebiological conduit along the previously positioned guide wire 58. One ormore radiopaque markers may optionally be provided on the device tomonitor the position of the device in the biological conduit. Anymaterial that prevents passage of electromagnetic radiation isconsidered radiopaque and could be used. Preferred radiopaque materialsinclude, but are not limited to, platinum, gold, or silver. Theradiopaque material can be coated on the surface of all or a part of thetip 40, on all or part of the splines 12, 14 or other actuators, on theguide wire 58, or on some combination of the foregoing structures.Alternatively, a ring of radiopaque material can be attached to the tip40. The device may optionally be provided with onboard imaging, such asintravascular ultrasound or optical coherence tomography. The tip of thedevice may optionally be provided with optics that are used to determinethe position of the device or characteristics of the surroundingbiological conduit.

When the device is at its desired position in the biological conduit,the operator uses mechanical connection 54 to retract the exteriorcatheter component 22 rearwardly away from the guide tip 40. In apreferred embodiment, as the exterior catheter component 22 is withdrawnfrom over the tissue penetrators 16, 18, the zipper track 56 of theexterior catheter component 22 is withdrawn over the rails 36, 38 of thepair of splines 12, 14. This movement releases the pair of splines 12,14 from their constrained, back-to-back configuration shown in FIG. 22,and allows the shape memory material of the splines 12, 14 to adopttheir unconstrained, bowed configurations shown in FIG. 23. As thesplines 12, 14 move to their unconstrained, bowed configurations, thesplines come into contact with the inner surface of the wall(s) of thebiological conduit and the tissue penetrators 16, 18 on the exteriorsurfaces 28, 30 of the splines 12, 14 are pressed into the interiorsurface of the biological conduit at the position of the device.

After the tissue penetrators 16, 18 have entered the desired layer ofthe wall of a biological conduit, a fluid can be delivered through thefluid delivery lumens 44, 46 in the central catheter component 20,through the fluid delivery conduits 32, 34 on the pair of splines 12,14, and through the tissue penetrators 16, 18. When the delivery of thefluid is complete, the operator uses the mechanical connection 54 tomove the exterior catheter component 22 (which may also be referred toas a shielding component) forward over the central catheter component 20and over the pair of splines 12, 14 toward the guide tip 40. As theexterior catheter component 22 moves forward over the pair of splines12, 14, the zipper track 56 on the interior of the exterior cathetercomponent 22 passes over the rails 36, 38 on the pair of splines 12, 14,causing the splines 12, 14 to move from their unconstrained, bowedconfiguration back to their constrained configuration. When the exteriorcatheter component 22 has been entirely advanced over the pair splines12, 14 and again engages with the guide tip 40, the zipper track 56 inthe exterior catheter component 22 holds the splines 12, 14 in theirconstrained configuration. The device then can be repositioned forrelease at another location in the biological conduit or anotherbiological conduit, or withdrawn from the body.

The shape and length of the splines 12, 14 are selected such thatvarious embodiments of the device can be used in biological spaces orconduits of various sizes or diameters. In certain embodiments, thesplines may be flat or rounded. Flat splines preferably have a widthranging from about 0.2 mm to about 20 mm, a height ranging from about0.2 mm to about 5 mm, and a length ranging from about 10 mm to about 200mm, depending on the particular application. Rounded splines preferablyhave a diameter ranging from about 0.2 mm to about 20 mm and a lengthranging from about 10 mm to about 200 mm, depending on the particularapplication. In specific embodiments, flat splines are 3.5 mm to 5 mm, 5mm to 10 mm, 10 mm to 15 mm, 15 mm to 20 mm in width, or any rangetherewithin 3.5 mm to 10 mm); 3.5 mm to 5 mm, 5 mm to 10 mm. 10 mm to 15mm, 15 mm to 20 mm in height, or any range therewithin 3.5 mm to 10 mm);and 10 mm to 20 mm, 20 mm to 40 mm, 40 mm to 80 mm, 80 mm to 120 mm, 120mm to 150 mm or 150 to 200 mm in length, or any range therewithin (e.g.,10 mm to 40 mm), or any permutation of the foregoing (e.g., a width of 5mm to 10 mm, a height or 3.5 to 5 mm, and a length of 20 to 40 mm). Inother embodiments, rounded splines are 3.5 mm to 5 mm, 5 mm to 10 mm, 10mm to 15 mm, 15 mm to 20 mm in diameter, or any range therewithin (e.g.,3.5 mm to 10 mm) and 10 mm to 20 mm, 20 mm to 40 mm, 40 mm to 80 mm, 80mm to 120 mm, 120 mm to 150 mm or 150 to 200 mm in length, or any rangetherewithin (e.g., 10 mm to 40 mm), or any permutation of the foregoing(e.g., a diameter of 5 mm to 10 mm and a length of 20 to 40 mm).

In a second specific embodiment, shown in FIG. 27, the device of thepresent invention is a fluid delivery catheter 110 comprising a centralcatheter component 112 having an elongate length with a longitudinalaxis 113, one or more (and preferably two) flexible, resilient actuatorsthat, in this specific embodiment, are formed as tissue penetratorpresentation tubes 114, 116 that extend from the distal portion of thecentral catheter component 112. At least a portion of the tissuepresentation tubes 114, 116 are movable between a constrainedconfiguration which is oriented substantially parallel to the centrallongitudinal axis 113 of the catheter assembly and an unconstrainedconfiguration which is oriented substantially non-parallel to thecentral longitudinal axis 113 of the catheter.

The catheter further comprises one or more (and preferably two)flexible, elongate tissue penetrators 118, 120 that extend through thetwo tissue penetrator presentation tubes 114, 116, and an exteriordeployment tube 122 that extends over portions of the lengths of thecentral catheter component 112, the tissue penetrator presentation tubes114, 116, and the middle rail 132.

The central catheter component 112 and the exterior deployment tube 122may be constructed of any materials suitable for constructing catheters.Examples of such materials include, but are not limited to, silicone,polyurethane, nylon, Dacron, and PEBAX™.

The tissue penetrators 118, 120 connect to respective hubs 166, 168(FIG. 31). One or more of the pair of tissue penetrators 118, 120preferably has a diameter of about 0.2 mm (33 gauge) to about 3.4 mm (10gauge), more preferably 0.8 mm to 1.3 mm (about 18 to 21 gauge). One ormore of the pair of tissue penetrators may have a standard bevel, ashort bevel or a true short bevel. The pair of tissue penetrators 118,120 are preferably constructed of materials that allow the tissuepenetrators to flex along their lengths. Examples of such materialsinclude, but are not limited to, nickel, aluminum, steel and alloysthereof. In a specific embodiment, the tissue penetrators areconstructed of nitinol. The full length of the tissue penetrators 118,120 can be constructed of a single material, or the distal ends (e.g.,the distal 1 mm to the distal 20 mm), including the tips 156, 158, ofthe tissue penetrators 118, 120 may be constructed of one material andconnected to the respective hubs 166, 168 via a tubing constructed of adifferent material, e.g., plastic.

One or more of the pair of tissue penetrator presentation tubes 114, 116is preferably constructed of a flexible, resilient material. Suchflexible, resilient material can be deformed, e.g., when the tissuepenetrator presentation tubes 114, 116 are in the straight, constrainedconfiguration of FIG. 27, but returns to its original shape when thedeformation force is removed, e.g., when the tissue penetratorpresentation tubes 114, 116 are in the curved, unconstrainedconfiguration shown in FIG. 28. Any such flexible, resilient materialcan be used, including but not limited to surgical steel, aluminum,polypropylene, olefinic materials, polyurethane and other syntheticrubber or plastic materials. The pair of tissue penetrator presentationtubes 114, 116 is most preferably constructed of a shape memorymaterial. Examples of such shape memory materials include, but are notlimited to, copper-zinc-aluminum-nickel alloys, copper-aluminum-nickelalloys, and nickel-titanium (NiTi) alloys. In a preferred embodiment,the shape memory material is nitinol.

The central catheter component 112 has a flexible elongate length withopposite proximal 124 and distal 126 ends (FIG. 27). The distal end 126of the central catheter component is formed as a guide tip that has anexterior shape configuration that will guide the distal end 126 througha biological conduit. A guide wire bore 128 within middle rail 132extends through the center of the central catheter 112 from the proximalend 124 to the distal end 126. The guide wire bore 128 receives aflexible, elongate guide wire 130 for sliding movement of the bore 128over the wire (FIG. 29). The guide wire 130 is used to guide thecatheter assembly through a biological conduit. In certain embodiments,the guide wire 130 has a solid core, e.g., stainless steel orsuperelastic nitinol. The guide wire may optionally be constructed ofradiopaque material, either in its entirety or at its distal portions(e.g., the most distal 1 mm to 25 mm or the most distal 1 mm to 3 mm).The guide wire 130 may optionally be coated with a medically inertcoating such as TEFLON®. In other embodiments, the catheter assembly isa rapid-exchange catheter assembly wherein a guide wire is positioned onthe distal end of the guide tip 126 and extends therefrom.

A narrow middle rail 132 surrounding the guide wire bore 128 extendsfrom the guide tip of the catheter distal end 126 toward the catheterproximal end 124. The middle rail 132 connects the guide tip 126 to abase portion 138 of the central catheter component.

The central catheter component base portion 138 has a cylindricalexterior surface that extends along the entire length of the baseportion. The base portion 138 extends along a majority of the overalllength of the central catheter component 112. As shown in FIG. 29, theguide wire bore 128 extends through the center of the central cathetercomponent base portion 138. In addition, a pair of tissue penetratorlumens 140, 142 also extend through the length of the central cathetercomponent base portion 138 alongside the guide wire bore 128. At theproximal end 124 of the central catheter component, a pair of ports 144,146 communicate the pair of lumens 140, 142 with the exterior of thecentral catheter component 112 (FIG. 27).

In an alternative embodiment, the medical device of FIG. 27 also maycomprise a single flexible, resilient actuator that is formed as atissue penetrator presentation tube, a single flexible, elongate tissuepenetrator that extends through the tissue penetrator presentation tubeand connects to a hub, and an exterior deployment tube that extends overportions of the lengths of the central catheter component, the tissuepenetrator presentation tube, and the middle rail.

The pair of first and second tissue penetrator presentation tubes 114,116 project from the catheter central component base portion 138 towardthe catheter distal end 126. Each of the tissue penetrator presentationtubes is formed as a narrow, elongate tube having a proximal end that issecured to the central catheter component base portion 138, and anopposite distal end 148, 150. Each of the first and second tissuepenetrator presentation tubes 114, 116 has an interior bore 152, 154that communicates with the respective first tissue penetrator lumen 140and second tissue penetrator lumen 142 in the central catheter componentbase portion 138.

As shown in FIGS. 29 and 30, the exterior configurations of the tissuepenetrator presentation tubes 114, 116 are matched to the middle rail132 so that the lengths of the tissue penetrator presentation tubes 114,116 may be positioned side-by-side on opposite sides of the middle rail132. The tissue penetrator tube distal ends 148, 150 can be formed asguide tip surfaces that also facilitate the passage of the catheterthrough a vascular system. The tissue penetrator tube distal ends 148,150 are preferably larger in diameter than the tissue penetratorpresentation tubes 114, 116. In a specific embodiment, the tissuepenetrator tube distal tips 148, 150 are rounded and bulbous tips. Suchtips are atraumatic and the tubes will not inadvertently puncture thewall of a biological conduit. The tips 148, 150 are exposed and do notextend outwardly beyond the diameter of the guide tip 126.

Each of the tissue penetrator tubes 114, 116 is preferably constructedof a shape memory material, such as nitinol. The tubes 114, 116 areformed with curved, unconstrained configurations shown in FIG. 28. Thetubes 114, 116 move to the curved, unconstrained configurations shown inFIG. 28 when no constraining force is applied against the tubes. Inorder for the presentation tubes 114, 116 to lie in straight,constrained configurations along the middle rail 132, a constrainingforce must be applied to the tubes to keep them in their straight,constrained positions shown in FIG. 27. As each of the tubes 114, 116moves from its straight, constrained configuration shown in FIG. 28 toits curved, unconstrained configuration shown in FIG. 28, the tissuepenetrator bores 152, 154 extending through the tubes also move fromstraight configurations to curved configurations.

The pair of tissue penetrators 118, 120, from their distal tips to thehubs 166, 168, have lengths that are slightly longer than the combinedlengths of the tissue penetrator lumens 140, 142 extending through thecentral catheter base portion 138 and the tissue penetrator bores 152,154 extending through the tissue penetrator presentation tubes 114, 116.The tips 156, 158 of the tissue penetrators 118, 120 are positionedadjacent to the distal ends 148, 150 of the tissue penetratorpresentation tubes 114, 116 and are positioned inside of the bores 152,154 of the tubes in the constrained configuration of FIG. 27. Theopposite, proximal ends of the tissue penetrators 118, 120 project outthrough the side ports 144, 146 of the central catheter 112. The pair oftissue penetrators 118, 120 are dimensioned to easily slide through thetissue penetrator lumens 140, 142 of the central catheter component 112and the tissue penetrator bores 152, 154 of the tissue penetratorpresentation tubes 114, 116. The side ports 144, 146 of the centralcatheter component 112 are preferably at 20° to 90° angles to thecentral catheter proximal end 124, most preferably at 30° to 60° anglesto the central catheter proximal end 124.

A pair of manual operator movement to linear movement controllers 162,164 can be connected to the proximal ends of the tissue penetrators 118,120 and can be secured to the central catheter ports 144, 146 (FIG. 31).The controllers 162, 164 can be constructed to convert operator movementinto controlled linear movement of the tissue penetrators 118, 120through the central catheter tissue penetrator lumens 140, 142 andthrough the tissue penetrator presentation tube bores 152, 154. In oneembodiment, there are rotating controllers 162, 164 that can be manuallymoved in one direction, such that the tissue penetrator injection tips156, 158 at the tissue penetrator distal ends can be adjustablypositioned to extend a desired length out from the tissue penetratortube bores 152, 154 at the tissue penetrator tube distal ends 148, 150.By rotating the controllers in the opposite direction, the tissuepenetrators 118, 120 can be retracted back into the tissue penetratortube bores 152, 154. Each of the operator movement to linear movementcontrollers 162, 164 can be provided with a hub 166, 168 thatcommunicates with the interior bore extending through the tissuepenetrators 118, 120 and can be used to connect a syringe or tubingcontaining a solution of a diagnostic or therapeutic agent.

The exterior deployment tube 122 has a tubular length that surrounds thecentral catheter 112, the tissue penetrator presentation tubes 114, 116,and the middle rail 132. The deployment tube 122 can be mounted on thecentral catheter component 112 and the pair of tissue penetratorpresentation tubes 114, 116 for sliding movement to a forward positionof the deployment tube 122 where an open distal end 172 of thedeployment tube is positioned adjacent the distal ends 148, 150 of thetissue penetrator presentation tubes 114, 116 as shown in FIG. 27, and arearward position of the deployment tube 122 where the tube distal end172 is positioned adjacent to the connection of the tissue penetratorpresentation tubes 114, 116 with the central catheter component 112 asshown in FIG. 28. The opposite proximal end 174 of the deployment tube122 can be provided with a mechanical connection 176 to the centralcatheter 112. The mechanical connection 176 enables the deployment tube122 to be moved between its forward and rearward positions relative tothe central catheter 112 and the tissue penetrator presentation tubes114, 116 (FIGS. 27 and 28). Such a connection could be provided by athumbwheel, a sliding connection, a trigger or push button or some otherconnection that is manually operable to cause the deployment tube 122 tomove relative to the central catheter 112 and the presentation tubes114, 116. When the deployment tube 122 is moved to its forward positionshown in FIG. 27, the tube distal end 172 passes over the lengths of thetissue penetrator presentation tubes 114, 116 and moves the presentationtubes to their constrained positions extending along the opposite sidesof the central catheter middle rail 132. When the deployment tube 122 ismoved to its rearward position shown in FIG. 28, the distal end 172 ofthe deployment tube is retracted from over the length of the tissuepenetrator presentation tubes 114, 116 and gradually allows thepresentation tubes 114, 116 to release their constrained energy and moveto their curved, unconstrained configurations shown in FIG. 28.

In use of the catheter 110, the deployment tube 122 is in the forwardposition shown in FIG. 27. The guide wire 130 is positioned in abiological conduit (such as an artery or vein) in a known manner. Thecatheter is then advanced into the biological conduit over the guidewire. The guide wire 130 extends from the biological conduit, and entersthe central catheter component distal end 126 through the guide wirelumen 128. The wire 130 passes through the length of the centralcatheter 112 and emerges at the proximal end of the central cathetercomponent adjacent to the catheter ports 144, 146, where the guide wire130 can be manually manipulated.

The catheter 110 can be advanced through the biological conduit and canbe guided by the guide wire 130. Radiopaque markers may optionally beprovided on the assembly to monitor the position of the assembly in thebiological conduit. Any material that prevents passage ofelectromagnetic radiation is considered radiopaque and may be used.Useful radiopaque materials include, but are not limited to, platinum,gold, or silver. The radiopaque material can be coated on the surface ofall or a part of the tip 126, on all or part of the presentation tubes114, 116, on all or part of the tissue penetrators 118, 120, on theguide wire 130, or on any combination of the foregoing structures.Alternatively, a ring of radiopaque material can be attached to the tip126. The assembly may optionally be provided with onboard imaging, suchas intravascular ultrasound or optical coherence tomography. The tip ofthe assembly may optionally be provided with optics that are useful fordetermining the position of the assembly or the characteristics of thesurrounding biological conduit. When the assembly is at a desiredposition, the exterior deployment tube 122 can be moved from its forwardposition shown in FIG. 27 toward its rearward position shown in FIG. 28by manual manipulation of the mechanical connection 176.

As the deployment tube 122 is withdrawn from over the pair of tissuepenetrator presentation tubes 114, 116, the constrained energy of thetissue penetrator presentation tubes 114, 116 is released and the tubesmove toward their unconstrained, curved configurations shown in FIG. 28.This movement positions the tissue penetrator bores 152, 154 at thetissue penetrator tube distal ends 148, 150 against the interiorsurfaces of the biological conduit into which the assembly 110 has beeninserted.

The operator movement to linear movement controllers 162, 164 then canbe manually operated to extend the tissue penetrator distal ends 156,158 from the tissue penetrator bores 152, 154 at the tissue penetratorpresentation tube distal ends 148, 150. A gauge may be provided on eachof the operator movement to linear movement controllers 162, 164 thatprovides a visual indication of the extent of the projection of thetissue penetrator tips 156, 158 from the tissue penetrator tube ends148, 150 as the controllers 162, 164 are rotated. The controllers alsocould provide an audible sound or tactile feel such as clicking toindicate incremental distance steps of the tissue penetrator movements.This deploys the tissue penetrator tips 156, 158 a desired distance intothe walls of the biological conduit.

In a third specific embodiment, a medical device of the instantinvention is a fluid delivery catheter comprising one or more tissuepenetrators constructed of a flexible, resilient material. In certainaspects, the medical device of the present invention has a centrallongitudinal axis, and comprises one or more tissue penetrators, whereinthe one or more tissue penetrators can exist in a constrainedconfiguration in which a length of said one or more tissue penetratorsis oriented substantially parallel to the longitudinal axis of saidmedical device and an unconstrained configuration in which at least aportion of the length of said one or more tissue penetrators is orientedsubstantially non-parallel to the device's central longitudinal axis.After the device is positioned at a target site adjacent to the wall ofa biological conduit, one or more tissue penetrators (and if desired,all of the tissue penetrators) may be released from a constrainedconfiguration and permitted to adopt an unconstrained configuration,thereby making contact with the wall of the biological conduit. The oneor more tissue penetrators may be of any shape, and in preferredembodiments, the movement of the one or more tissue penetrators from theconstrained configuration to the unconstrained configuration occurs uponrelease of a constraining force by the device operator but without theinput by the operator of any deforming forces to the device or thetarget tissue.

In a preferred embodiment, tissue penetrators are constructed offlexible, resilient material that is capable of being constrained uponthe application of a constraining force, e.g., when the tissuepenetrators are in the constrained configuration, and adopts itsoriginal unconstrained shape when the constraining force is removed,e.g., when the tissue penetrators are in the unconstrainedconfiguration. Any such flexible, resilient material can be used,including but not limited to surgical steel, aluminum, polypropylene,olefinic materials, polyurethane and other synthetic rubber or plasticmaterials. The one or more tissue penetrators are most preferablyconstructed of a shape memory material. Examples of such shape memorymaterials include, but are not limited to, copper-zinc-aluminum-nickelalloys, copper-aluminum-nickel alloys, and nickel-titanium (NiTi)alloys. In a preferred embodiment, the shape memory material is nitinol.In a preferred embodiment, when the tissue penetrators assume theunconstrained configuration, the shape memory properties of the materialfrom which each tissue penetrator is formed cause the tissuepenetrators, without the application of any external deforming force, tomove from a position substantially parallel to the longitudinal axis ofthe medical device to a position substantially perpendicular to thelongitudinal axis of the medical device.

In a preferred embodiment, the tissue penetrators are maintained in theconstrained configuration by an exterior catheter component having atubular configuration that surrounds the tissue penetrators. Amechanical connection is provided between the exterior cathetercomponent and the central catheter component to which the tissuepenetrators are attached. The mechanical connection enables the exteriorcatheter component to be moved rearwardly along the length of thecentral catheter component, thereby uncovering the constrained one ormore tissue penetrators and permitting the one or more tissuepenetrators to assume an unconstrained configuration wherein they makecontact with the target delivery site. One of ordinary skill in the artwould appreciate that this specific embodiment may be readily adapted toincorporate radiopaque markers to facilitate positioning of the deviceor rapid-exchange features to facilitate the use of the device.

The medical device of the present invention, in its various embodiments,permits delivery of fluids into distinct layers of a vascular wall. Thevascular wall consists of numerous structures and layers, structures andlayers, including the endothelial layer and the basement membrane layer(collectively the intimal layer), the internal elastic lamina, themedial layer, and the adventitial layer. These layers are arranged suchthat the endothelium is exposed to the lumen of the vessel and thebasement membrane, the intima, the internal elastic lamina, the media,and the adventitia are each successively layered over the endothelium asdescribed in U.S. Pat. App. Publication No. 2006/0189941A1. With themedical devices of the present invention, the depth to which the tissuepenetrator tips 156, 158 can penetrate into the target tissue can becontrolled by rotating the controllers 162, 164. For example, if thetarget layer is the adventitial layer, the constrained energy of thetubes 114, 116 is released, the tubes adopt their unconstrained, curvedconfigurations shown in FIG. 28, and the tissue penetrator tips 156, 158are advanced with the controllers to a length sufficient for penetrationto the depth of the adventitial layer. Likewise, if the target layer isthe medial layer, the constrained energy of the tubes 114, 116 isreleased, the tubes adopt their unconstrained, curved configurationsshown in FIG. 28, and the tissue penetrator tips 156, 158 are advancedwith the controllers to a length sufficient for penetration to the depthof the medial layer.

With the tissue penetrators embedded in the desired layer of the wall ofthe biological conduit, a fluid can then be delivered through the tissuepenetrators 118, 120. When the delivery of the fluid is complete, thecontrollers 162, 164 can be operated to withdraw the tissue penetratortips 156, 158 back into the interior bores 152, 154 of the tissuepenetrator presentation tubes 114, 116. The deployment tube 122 can thenbe moved to its forward position where the deployment tube distal end172 moves the tissue penetrator presentation tubes 114, 116 back totheir constrained positions shown in FIG. 27. When the deployment tube122 has been moved to its full forward position shown in FIG. 27, theassembly can then be repositioned or withdrawn from the body.

The medical device of the instant invention also permits delivery offluids to plaque deposits on the inside of the wall of the biologicalconduit or within the wall of the biological conduit.

The medical device of the instant invention also permits delivery offluids to extracellular spaces or tissues located outside of the outerwall of the biological conduit (e.g., to the exterior surface of a bloodvessel or to muscle positioned against the outer surface of vessel suchas myocardium).

One advantageous feature of the devices of the present invention is thatthe actuators, by virtue of their design, make contact with less thanthe complete circumference of the inner wall of a biological conduitfollowing their deployment therein. In preferred embodiments, theactuators make contact with less than 100% of the circumference of theinner wall of a biological conduit in which they are deployed. Morepreferably, the actuators make contact with less than 75%, 50% or 25% ofthe circumference of the inner wall of a biological conduit in whichthey are deployed. Most preferably, the actuators make contact with lessthan 10%, 5%, 2.5%, 1%, 0.5% or 0.1% of the circumference of the innerwall of a biological conduit in which they are deployed.

The devices can be used to deliver fluids comprising a variety oftherapeutic and/or diagnostic agents to a wall of a biological conduit.Therapeutic agents include, but are not limited to proteins, chemicals,small molecules, cells and nucleic acids. A therapeutic agent deliveredby the device may either comprise a microparticle or a nanoparticle, becomplexed with a microparticle or a nanoparticle, or be bound to amicroparticle or a nanoparticle. Protein agents include elastases,antiproliferative agents, and agents that inhibit vasospasm. The use ofthe devices for delivery of an elastase is specifically contemplated.Several published patent applications (WO 2001/21574; WO 2004/073504;and WO 2006/036804) teach that elastase, alone and in combination withother agents, is beneficial in the treatment of diseases of biologicalconduits, including obstruction of biological conduits and vasospasm.Diagnostic agents include, but are not limited to, contrast,microparticles, nanoparticles or other imaging agents.

A variety of distinct fluid delivery methods can be practiced with thedevice. In certain applications, distinct fluids can be deliveredthrough each tissue penetrator of the device either simultaneously orsequentially. In other applications, the same fluid can be deliveredthrough both tissue penetrators either simultaneously or sequentially.Embodiments and/or methods where a first fluid is delivered through bothtissue penetrators followed by delivery of a second fluid through bothtissue penetrators are also contemplated.

Methods of using the devices to deliver fluids into or through a wall ofa biological conduit are also specifically contemplated. These methodscomprise the steps of introducing the device into the biologicalconduit, advancing the device to a target site within the conduit,releasing the actuators from their constrained positions, optionallyadvancing the tissue penetrators through lumens in the actuators topenetrate to a desired depth into the wall of a biological conduit,delivering at least one fluid into or through the wall, optionallyreturning the tissue penetrators back into the lumens of the actuators,retracting the actuators to their constrained position, repositioningthe device in the same or a different conduit for the delivery ofadditional fluid if so desired, and removing the device from theconduit.

6. EXAMPLES

This section describes methods of production of recombinant type Ielastase for clinical use, for example as an agent to enlarge thediameter of blood vessels and thereby the lumen of blood vessels. Humantype I pancreatic elastase displays 89% amino acid identity across theentire length of the porcine type I pancreatic elastase, with completeconservation of the “catalytic triad” and substrate specificitydetermining residues. Porcine type I elastase is initially synthesizedas an enzymatically inactive proenzyme that is activated by trypsin toyield the mature enzyme that contains four internal disulfide bonds andno glycosylation (see Shotton, 1970, Methods Enzymol 19:113-140,Elastase; and Hartley and Shotton, 1971, Biochem. J. 124(2): 289-299,Pancreatic Elastase. The Enzymes 3:323-373 and references therein).

The examples below demonstrate the development of efficient and scalablerecombinant porcine and human type I elastase expression andpurification schemes suitable for cGMP manufacture of these enzymes fornon-clinical and clinical studies, and commercial pharmaceutical use.The mature porcine elastase has been given the name PRT-102. The maturehuman type I elastase has been given the name PRT-201.

6.1 Terminology and Abbreviations

As used herein, the terms PRT-101, PRT-102, PRT-201 and pro-PRT-201shall mean the following:

PRT-101: porcine pancreatic elastase. Unless otherwise indicated, theporcine pancreatic elastase employed in the examples is highly purifiedporcine pancreatic elastase purchased from Elastin Products Company,Inc, Owensville, Mo., catalog # EC134.

PRT-102: mature recombinant type I porcine pancreatic elastase. Itshould be noted that vectors with the designation “pPROT101-XXX” encodePRT-102.

PRT-201: mature recombinant type I human pancreatic elastase.

pro-PRT-201: proenzyme form of recombinant human type I pancreaticelastase containing a propeptide sequence.

The following abbreviations are used in Section 6 of the application:

BKGY: buffered glycerol-complex medium

BKME: buffered methanol-complex medium

CHO: Chinese hamster ovary

E. coli: Escherichia coli

ELA-1: type I pancreatic elastase

HEK: human embryonic kidney

hELA-1: human ELA-1

HIC: hydrophobic interaction chromatography

MBP: maltose binding protein

pELA-1: porcine ELA-1

P. pastoris: Pichia pastoris

PCR: polymerase chain reaction

PMSF: phenylmethylsulphonyl fluoride

RP: reversed phase

S. cerevisiae: Saccharomyces cerevisiae

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEC: size exclusion chromatography

USP: United States Pharmacopeia

YPDS: yeast extract peptone dextrose sorbitol medium

6.2 Example 1 Elastase DNA Synthesis

The human elastase-1 coding sequence was obtained from U.S. Pat. No.5,162,205 (Takiguichi et al., 1992). Several sequence changes were madeto facilitate cloning into expression vectors (FIG. 1A). The basechanges fell within the degeneracy of the genetic code so that no aminoacid residues were changed. A second stop codon was added immediatelyafter the native stop codon to minimize potential ribosome read through.

The modified coding sequence was synthesized by Blue Heron Biotechnology(Bothell, Wash.) using a non-PCR “long oligo” technique under licensefrom Amgen (Thousand Oaks, Calif.). The recombinant DNA, named ELA-1.2A(SEQ ID NO:81), was cloned into the vector Blue Heron pUC, a derivativeof pUC119, and the resulting plasmid was named pPROT1. pPROT1 wassequenced on both strands to confirm the correct sequence. High qualitysequencing data with extensive overlap on both strands permittedunambiguous base assignments across the entire sequence, which wascovered by a minimum of four sequencing reactions with at least onereaction for each strand.

6.3 Example 2 Expression of PRT-201 in E. Coli

A variety of expression strategies were attempted in an effort to obtainsoluble and enzymatically active human elastase in E. coli.

One set of E. coli expression vectors comprised in frame fusions ofhuman type I pancreatic elastase (ELA-1) to the carboxy terminus of aMaltose Binding Protein (MBP) that was in turn fused to an N-terminalsecretory peptide. Both the human ELA-1 mature and proenzyme codingsequences were cloned as in-frame C-terminal fusions to the MBP codingsequence of plasmid pMAL-p2G (New England Biolabs, Inc., Beverly, Mass.)to yield either pPROT3 (mature ELA-1) or pPROT5 (ELA-1 proenzyme).Construction of pPROT3 was effected by first obtaining by PCRmutagenesis a 6.6 kb mature human ELA-1 encoding fragment with a SnaBIsite at the N-terminal valine codon of mature human ELA-1 and a HindIIIsite located 3′ to the mature ELA-1 termination codons. This PCRmutagenesis used a pPROT1 template comprising the ELA-1.2A codingsequence (SEQ ID NO:81) and two oligonucleotide primers (5′ ATC TAC GTAGTC GGA GGG ACT GAG GCC, SEQ ID NO:75; and 5′ gtc gac aag ctt atc agttgg agg cga t, SEQ ID NO:76). The resultant 6.6 kb PCR fragment wasisolated, digested with SnaBI and HindIII, and subsequently cloned intothe XmaI/HindIII digested pMAL-p2G vector to yield pPROT3. Constructionof pPROT5 was effected by cloning the ScaI/HindIII fragment from pPROT1and ligating into the pMAL p2G vector that had been digested with SnaBIand HindIII. The resultant fusion operably links the N-terminus of thehuman ELA-1 proenzyme coding region to the C-terminus of the MBP ofpMAL-p2G in pPROT5. The trypsin cleavage domain of the human ELA-1proprotein is preserved in pPROT5.

E. coli strain TB 1 was transformed with pPROT3 and pPROT5 andsubsequently induced with IPTG to determine if either the fusion proteinor enzymatically active human ELA-1 could be produced. In the case ofpPROT3, all of the MBP-ELA-1 fusion protein produced was insoluble. Nosoluble or enzymatically active MBP-ELA-1 fusion protein was detected inthe periplasmic material obtained by osmotic shock of inducedpPROT3-containing E. coli. In the case of pPROT5, low levels of solublerecombinant MBP-proELA-1 protein could be detected in the periplasmicmaterial obtained by osmotic shock of induced pPROT5-containing E. colithrough use of SDS-PAGE and Coomassie stain or by use of anti-MBPantibodies on a Western blot (New England Biolabs, Inc., Beverly,Mass.). The soluble recombinant MBP-proELA-1 protein could be digestedwith trypsin to yield both MBP and mature human ELA-1. Mature humanELA-1 obtained from inductions of pPROT5 was subsequently assayed forelastase activity with SLAP peptide substrate. Elastase activity wasobserved in pPROT5 periplasmic extracts. This elastase enzymaticactivity was dependent on trypsin activation (i.e., no activity wasobserved in absence of trypsin and amount of activity is increased byincreasing the time period of trypsin activation). Moreover, theelastase activity was dependent on pPROT5 in as much as no activity wasobserved in pMAL-p2G vector control extracts treated in parallel withtrypsin. Finally, the pPROT5 elastase activity was inhibited by PMSF (aknown serine protease inhibitor).

The recombinant MBP-proELA-1 fusion protein was subsequently purifiedand cleaved with trypsin to obtain an enzymatically activepPROT5-derived mature human ELA-1. The fusion protein was first purifiedon amylose affinity chromatography followed by elution with maltose. Thepurified MBP-proELA-1 was then treated with immobilized trypsin.Following the trypsin activation step, the cleaved MBP-proELA-1 waspurified by cationic SP Sepharose chromatography. However, subsequentexperiments with affinity purified pPROT5-derived mature human ELA-1indicated that only very limited amounts of soluble and enzymaticallyactive elastase could be obtained from E. coli containing pPROT5.Moreover, the specific activity of the affinity purified, pPROT5-derivedmature human ELA-1 was very low, ranging from 0.27 to 0.38 U/mg(U=micromole of SLAP substrate hydrolyzed per min).

An alternative strategy of obtaining soluble and enzymatically activeELA-1 in E. coli was also pursued. In brief, the pPROT8 vector thatencodes a protein fusion comprising the first 8 amino acid residues ofthe E. coli lacZ alpha subunit plus 5 amino acids of poly linker encodedresidues followed by the human ELA-1 N-terminal proenzyme wasconstructed. This vector was constructed by ligating a BamHI/NcoIfragment containing the human ELA-1 coding sequence from pPROT1 (i.e., avector containing the ELA-1.2A sequence; SEQ ID NO: 81) into pBlueHeronpUC (Blue Heron Biotechnology, Bothell, Wash., USA) that was digestedwith BamHI/NcoI.

E. coli strain EC100 (EPICENTRE Biotechnologies, Madison, Wis.) wastransformed with pPROT8 and subsequently induced with IPTG to determineif either the fusion protein or enzymatically active human ELA-1 couldbe produced. In the case of EC100 transformed cells, all of the pPROT8derived LacZ-proELA-1 fusion protein produced was insoluble (i.e., foundin inclusion bodies). An E. coli strain containing mutations in the trxBand gor genes (the Origami™ strain, Takara Minis Bio, Inc., Madison,Wis.) was subsequently transformed with pPROT8 as E. coli strains withmutations in these coding sequences are known to promote recovery ofsoluble and enzymatically active recombinant proteins. Although somesoluble pPROT8 derived LacZ-proELA-1 fusion protein in the trxB/gor E.coli strain was recovered upon induction with IPTG, it could not beconverted to enzymatically active human ELA-1 with trypsin.

6.4 Example 3 Expression of PRT-201 in Mammalian Cell Lines

Several expression strategies were attempted in an effort to obtainsoluble and enzymatically active human type I pancreatic elastase(ELA-1) in mammalian cell lines. The high copy mammalian expressionvector pcDNA3.1 (Invitrogen) containing the CMV promoter was used as abackbone for two human ELA-1 elastase expression vectors, pPROT30 andpPROT31. To construct pPROT30, the human ELA-1 proenzyme sequence wasamplified by PCR and fused to a porcine pancreatic elastase signalsequence incorporated in the forward PCR primer. Using restriction sitesincorporated into the PCR primers, the PCR product was digested andligated using the corresponding restriction sites in the pcDNA3.1vector. pPROT31 was constructed in a similar fashion, except that thehuman ELA-1 mature coding sequence was used instead of the proenzymecoding sequence in an attempt at direct expression of the mature enzyme.E. coli was transformed with the ligation reactions and clones wereselected for miniprep screening. One clone for each expression vectorwas selected based on expected restriction digest patterns for thecorrect insert. Plasmid DNA was prepared for each clone and theexpression vector coding sequences were confirmed by DNA sequencing.

The mammalian cell lines CHO, COS, HEK293 and HEK293T were transientlytransfected separately with pPROT30 and pPROT31. After several days,cell culture supernatants were harvested and analyzed for human ELA-1proprotein (pPROT30) or mature protein (pPROT31) expression by Westernblot. An anti-porcine pancreatic elastase polyclonal antibodycross-reacted with a band of the expected molecular weight for the humanELA-1 proprotein in pPROT30 supernatants and for the mature human ELA-1protein in pPROT31 supernatants.

The pPROT30 and pPROT31 supernatants were analyzed for elastase activityby SLAP assay. For pPROT30, the supernatants were first treated withtrypsin to convert the proenzyme to mature PRT-201. No elastase activitywas detected in any of the supernatants for either vector using the SLAPassay.

6.5 Example 4 Expression of Trypsin-Activated PRT-201 in P. pastoris

The vector for P. pastoris secreted expression, PV-1, was synthesized byBlue Heron and used to first clone the wild-type human ELA-1 codingsequence. The PV-1 vector was designed for simple cloning, selection andhigh-level expression of the recombinant protein. The vector containsthe Zeocin™ resistance gene for direct selection of multi-copyintegrants. Fusion of the N-terminus of the elastase propeptide to ayeast α-mating type sequence comprising the yeast secretion signal,propeptide and spacer sequences as shown in FIG. 1B permits secretion ofthe expressed protein in the culture media. The secreted elastaseproprotein can be easily separated from the cell pellet, a substantialfirst step towards purification. Additionally, the proenzyme form ofhuman ELA-1 containing the trypsin cleavage site was selected forexpression to avoid directly expressing the mature, activated enzymewhich may lead to protein misfolding or toxicity to the cells expressingthe recombinant enzyme.

Cloning of the expression construct pPROT24-V to direct the expressionof ELA-1 proenzyme was accomplished as follows. The human ELA-1 codingregion (SEQ ID NO:81) was amplified from Blue Heron pUC ELA-1 by PCR(Expand High Fidelity PCR System, Roche, Indianapolis, Ind.). The 20Fforward primer incorporated an XhoI site(5′-ggctcgagaaaagagaggctgaagctactcaggaccttccggaaaccaatgcccgg-3; SEQ IDNO:35). The 24R primer incorporated a SacII site(5′-gggccgcggcttatcagttggaggcgatgacat-3′; SEQ ID NO:36). The resultingPCR product was gel-purified and cloned into pCR2.1-TOPO (Invitrogen).The ELA-1 coding sequence was isolated using XhoI and SacII,gel-purified, and cloned into PV-1 vector at those sites to yieldpPROT24-V (FIG. 3).

The pPROT24-V ligated product was amplified in E. coli strain TOP10. Thecell mixture was plated on low salt LB plates supplemented with 25microgram/mL Zeocin. DNA plasmid was prepared (Qiagen, Valencia, Calif.)and the human ELA-1 insert was identified by restriction digestion. ThepPROT24 coding sequence was verified by sequencing both strands ofpurified maxiprep DNA with multiple overlapping reactions. High qualitysequencing data allowed unambiguous base assignments and confirmed thecorrect coding sequence. Glycerol stocks of pPROT24-V/TOP10 were madeand stored at −80° C.

The wild-type NRRL Y-11430 P. pastoris strain obtained from the UnitedStates Department of Agriculture (USDA, Peoria, Ill., USA) was used fortransformation. Plasmid DNA from pPROT24-V was linearized with SacI andcomplete digestion was confirmed by running a small aliquot of thereaction on an agarose gel. Electroporation was used to transform P.pastoris with pPROT24-V plasmid DNA. Cell mixtures were plated onto YPDSplates containing 100 microgram/mL Zeocin. After three days, coloniesbegan to form and were selected over several more days for re-streakingon fresh plates.

In general, drug-resistant transformants were screened for expression ina 1 L baffled flask. A single colony was used to inoculate 200 mL ofBKGY medium. The composition of the BKGY solution was the following: 10g/L glycerol, 13.4 g/L yeast nitrogen base with ammonium sulfate andwithout amino acids (Invitrogen), 20 g/L soy peptone, 10 g/L yeastextract, 0.4 mg/L biotin in 0.1 M potassium-phosphate buffer (pH 5.0).The culture was grown for two days at 28° C. with shaking at 275 rpm.The cultures were pelleted by centrifugation at 650×g for 10 min at roomtemperature. The cell pellets were resuspended with BKME inductionmedia, pH 5.0, by resuspending the pellets at a ratio of 1 g wet cellsto 5 mL induction media. A 50 mL cell suspension was placed in a 500 mLnon-baffled flask to obtain a 1:10 ratio of cell suspension to flaskvolume. The cells were incubated at 22° C. with shaking at 275 rpm for1-3 days. Methanol in the induction media was replenished to a finalconcentration of 0.5% twice daily over the course of induction.

To screen for expression, 1 mL aliquots were taken, transferred to 1.5mL microfuge tubes and centrifuged for 5 min at 20,000×g in amicrocentrifuge. Supernatants were transferred to fresh tubes and storedat −80° C. For SDS-PAGE analysis, supernatant aliquots were thawed andmixed with 4× Laemmli buffer supplemented to 5% volume with the reducingagent beta-mercaptoethanol. Samples were boiled for 5 min, gentlycentrifuged, and loaded onto an 8-16% gradient Tris-HCl pre-cast gel ina Criterion electrophoresis system (Bio-Rad). After electrophoresis, thegel was stained with Coomassie and analyzed for human ELA-1 proenzymeexpression. Clone 201-24-266-VU was selected as a high-yield clone forfurther evaluation. A development cell bank consisting of 201-24-266-VUglycerol stocks was prepared and stored at −80° C.

For scale-up production of human ELA-1 proenzyme using clone201-24-266-VU, multiple production runs were performed that generallyfollowed the methods described below. Where applicable, run-to-runvariations in the methods are noted.

For cell culture, a series of 2 L baffled shaker flasks (typicallyranging from 20 to 40 flasks) containing 500 mL BKGY growth medium wereinoculated with 250 microliters of thawed 201-24-266-VU glycerol stock.Cultures were grown at 28° C. for 2 days in a shaking incubator at250-300 rpm. After 2 days, the cells were pelleted by centrifugation andthe supernatant was discarded. The cells were resuspended in BKMEinduction media, pH 5.0, at a ratio of 1 g of weight cells to 5 mLmedia. A volume of 200-400 mL cell suspension was placed in 2 Lnon-baffled flasks and cultured for 3 days in a shaking incubator(250-300 rpm) at 22° C. Methanol in the media was replenished to 0.5%volume twice daily during the course of induction. At the end ofinduction, the shake flask cultures were centrifuged to pellet thecells. The supernatant was removed and immediately filtered at roomtemperature through a 0.22 um polyethersufone membrane using 1 L vacuumfiltration units to remove any remaining cell debris. The filtrate wasstored at 2-8° C. for up to 1.5 months. Based on HIC-HPLC analysis, theyield of human ELA-1 proenzyme from clone pro-PRT-201-24-266-VU in theclarified supernatant was typically 200 to 250 mg/L.

Capture of pro-PRT-201-24-266-VU from the supernatant was effected asfollows. First, supernatant from multiple rounds of shaker flaskcultures (typically 4 to 10 rounds) was combined (typically 8 to 25 Ltotal), diluted 8-fold with water and adjusted to pH 5.0 with 1 M HCl.The diluted supernatant was then loaded onto an equilibrated 2 L bedvolume Macro-Prep High S ionic exchange capture column at 2-8° C. at arate of 100 mL/min (linear flow rate 76 cm/hr). The chromatographyprogram comprised the following steps: 1. Wash column with 10 L (5column volumes [CVs]) of Buffer A (20 mM sodium citrate, pH 5.0) at 100mL/min (76 cm/hr); 2. Wash column with 4 L (2 CVs) of a mixture of 90%Buffer A and 10% Buffer B (500 mM sodium chloride; 20 mM sodium citrate,pH 5.0) for a final buffer composition of 50 mM sodium chloride; 20 mMsodium citrate, pH 5.0 at 100 mL/min (76 cm/hr); 3. Wash column with 6 L(3 CVs) of a mixture of 80% Buffer A and 20% Buffer B for a final buffercomposition of 100 mM sodium chloride; 20 mM sodium citrate, pH 5.0 at100 ml/min (76 cm/hr); 4. Wash column with 6 L (3 CVs) of a lineargradient, starting from 75% Buffer A and 25% Buffer B to 68% Buffer Aand 32% Buffer B, at 100 ml/min (153 cm/hr); 5. Elute with a lineargradient of 30 L (15 CVs) starting from 68% Buffer A and 32% Buffer B to0% Buffer A and 100% Buffer B, at 100 ml/min (76 cm/hr). The eluate wascollected in fractions of 500-1000 mL each.

Typically, two predominant protein species were observed by SDS-PAGEfollowed by Coomassie staining: the glycosylated human ELA-1 proenzymeand the non-glycosylated human ELA-1 proenzyme, as determined bysubsequent LC/MS analysis, typically eluting at about 320 mM sodiumchloride, shown in FIG. 4. In some production runs a protein slightlysmaller than the human ELA-1 proenzyme was observed as a minor species.Subsequent LC/MS analysis of these fractions showed that most of theprotein was not full-length proenzyme but instead was lacking severalamino acids at the N-terminus. These N-terminal variants were purifiedand subjected to elastase activity analysis, which revealed that theyhad lower elastase activity than full length PRT-201. N-terminalvariants could arise from the human ELA-1 proenzyme exhibiting a lowlevel of elastase activity during cell culture and capturechromatography operations and either cleaving itself through anintramolecular reaction or cleaving another human ELA-1 proenzymemolecule through an intermolecular reaction. Suboptimal cleavageconditions during these operations could lead to a high level ofinaccurate cleavage of the proenzyme (sometimes referred to asspontaneous or uncontrolled conversion) resulting in mostly N-terminalvariants, rather than intact, full length PRT-201.

After inspection of SDS-PAGE results, a subset of fractions was pooledto obtain purified non-glycosylated pro-PRT-201 for further processing.Fractions containing glycosylated proenzyme or full-length PRT-201and/or N-terminal variants (which co-migrate on SDS-PAGE) were typicallyexcluded from the pooling. The pooled pro-PRT-201 was typically storedin a 5 L plastic beaker at 2-8° C. for several hours to overnight priorto conversion from proenzyme to mature enzyme.

Conversion of pro-PRT-201 to mature PRT-201 by immobilized trypsin waseffected as follows. The pooled pro-PRT-201 fractions were dialyzed in20 mM sodium phosphate, pH 5.0, overnight at 2-8° C. This step providesfor the removal of citrate, which inhibits trypsin. Following dialysis,the pro-PRT-201 was passed over a column of recombinant trypsin(TrypZean) immobilized to agarose beads pre-equilibrated with 20 mMsodium phosphate, pH 5.0. Typically, the contact time betweenpro-PRT-201 and the immobilized TrypZean was between 3.5 to 5 min. Theresulting post-conversion material was analyzed by SDS-PAGE to confirmconversion and subsequently loaded onto a Macro-Prep High S polishcolumn. The column was washed sequentially with 5 CVs of 20 mM sodiumcitrate, pH 5.0; 2 CVs of 20 mM sodium citrate, 50 mM sodium chloride,pH 5.0; and 3 CVs of 20 mM sodium citrate, 100 mM sodium chloride, pH5.0. The column was further washed with a linear gradient from 125 mMsodium chloride to 160 mM sodium chloride in 20 mM sodium citrate, pH5.0. PRT-201 was eluted in a linear gradient from 165 mM to 500 mMsodium chloride in 20 mM sodium citrate, pH 5.0 and collected infractions. Fractions were analyzed for protein by SDS-PAGE followed byCoomassie staining and for elastase activity by SLAP assay. Typically,fractions having a specific activity of 90% or greater than that of thefraction with the highest specific activity were pooled. In subsequentLC/MS analysis, late-eluting fractions having lower specific activitywere found to be enriched in PRT-201 N-terminal variants.

Pooled fractions with high specific activity were diafiltered intoformulation buffer composed of 0.1×PBS (13.7 mM sodium chloride, 1 mMsodium phosphate, 0.27 mM potassium phosphate) at pH 5.0. Afterconcentration of PRT-201 to 1 mg/mL, the pH was adjusted to 7.4. Thesolution was then aliquoted into glass serum vials with an elastomerstopper, and lyophilized. Lyophilization was typically performed with aprimary drying cycle at −30 to −50° C. and a secondary drying cycle at−15° C. After lyophilization, the vials were stoppered under vacuum andcrimped with an aluminum seal. The vials were then typically stored at2-8° C. or −80° C.

To evaluate the stability of lyophilized PRT-201 in vials, two lots thatwere manufactured into sterile GMP drug product have been placed on astability program. The stability program consists of storage of the drugproduct at −15° C. and periodic removal of a subset of vials for testingby the following stability-indicating analytical methods (specificationsare in parentheses): appearance of lyophilized material (white tooff-white powder), appearance after reconstitution (clear, colorlesssolution free of particles), specific activity by SLAP assay (25-45U/mg), purity by RP-HPLC (total purity: not less than 93%; individualimpurity: not more than 2%), purity by reduced SDS-PAGE (not less than93%), purity by non-reduced SDS-PAGE (not less than 93%), particulatematter injections (conforms to USP), aggregates by SEC-HPLC (not morethan 3%), content per vial (4.5-5.5 mg), pH (6.5-8.5), moisture (doesnot exceed 5%) and sterility (conforms to USP). To date, both lots havemet the indicated specifications at all time points tested (lot C0807117through 12 months and lot C1007132 through 9 months). These resultsindicate that PRT-201 is stable for at least 12 months when storedlyophilized in vials at −15° C.

The use of sodium citrate, pH 5.0, during both capture chromatography ofpro-PRT-201 and polish chromatography of converted PRT-201 was based ondata showing that sodium citrate inhibited elastase activity of purifiedPRT-201 and therefore might also inhibit elastase activity ofpro-PRT-201 and PRT-201 during processing operations. Such inhibitioncould minimize the spontaneous conversion of pro-PRT-201 to N-terminalvariants and minimize auto-degradation of PRT-201. In an experimentwhere SLAP assay buffer contained 115 mM sodium citrate, pH 5.0, thespecific activity of PRT-201 was inhibited by 91%.

Occasionally, eluted material from the pro-PRT-201 capture column wasnot subjected to conversion by trypsin as described above but insteadused to obtain preparations enriched in various protein species. Forexample, pools of fractions containing primarily glycosylatedpro-PRT-201, non-glycosylated pro-PRT-201 or proteins arising fromspontaneous conversion were made from the capture column eluate.Typically, these fraction pools were diafiltered into 10 mM sodiumphosphate, pH 5.0, lyophilized, and stored at −80° C.

A study was performed to determine the effect of pH and temperature onthe stability of purified pro-PRT-201 over time. Lyophilized pro-PRT-201was reconstituted in 10 mM sodium phosphate at pHs ranging from 3.0 to8.4, followed by incubation at 4° C. or 25° C. for 7 days. After 7 days,the pro-PRT-201 samples under conditions of pH 4.0-8.0 and 25° C. showedan enrichment in mature PRT-201 that increased with increasing pH asshown by SDS-PAGE and Coomassie staining. Under the conditions of pH 8.4and 25° C., complete conversion of pro-PRT-201 to mature PRT-201 wasobserved. The samples at 4° C., from pH 4.0 to 8.4 showed little to noconversion. At pH 3.0, no conversion was observed at either 4° C. and25° C. It has been reported in the literature (Hartley and Shotton,1971, Pancreatic Elastase. Enzymes 3:323-373) that porcine pancreaticelastase is irreversibly inactived after prolonged storage at pHs lowerthan 3.0. Thus, based these results of this study and to avoidirreversibly inactivating PRT-201, useful conditions to minimizeconversion of purified pro-PRT-201 are storage at 4° C. between pH3.0-4.0.

6.6 Example 5 Expression of Auto-Activated PRT-201 in P. pastoris

To obtain a variant proenzyme capable of auto-activation, therebyeliminating the need for trypsin activation, a variety of elastasecleavage domain variant vectors were constructed and analyzed insmall-scale culture and conversion experiments. The variant vectors werecreated by site-directed PCR mutagenesis of the pPROT24-V vector andsubsequent derivative vectors. Site-directed mutagenesis was performedusing Pfu Turbo DNA polymerase (Stratagene). The E. coli XL10-Goldstrain was transformed with the resulting plasmids. The transformed cellmixtures were plated on low salt LB plates supplemented with 25microgram/mL Zeocin. Drug-resistant clones were picked and plasmid DNAwas prepared (Qiagen, Valencia, Calif.). Clones were confirmed for theexpected codon changes by sequencing both strands of plasmid DNA in thepropeptide region with multiple overlapping reactions. P. pastoris wastransformed with the variant vectors and clones were selected asdescribed in the preceding Example.

A summary of the elastase cleavage domain variants that were created isprovided in Table 4 below:

TABLE 4 Elastase cleavage domain variants Pro- peptide Cleaved sequencebond name P5 P4 P3 P2 P1 P′1 P′2 P′3 24 Glu Thr Asn Ala Arg Val Val Gly40 Glu Thr Ala Ala Ala Val Val Gly 41 Glu Thr Asn Ala Ala Ala Val Gly 42Glu Thr Asn Ala Ala Val Val Gly 43 Glu Thr Asn Ala Pro Val Val Gly 44Glu Thr Gly Ala Gly Ile Val Gly 45 Glu Thr Val Pro Gly Val Val Gly 46Glu Thr Ala Pro Gly Val Val Gly 47 Glu Thr Asn Pro Gly Val Val Gly 48Glu Thr Asn Pro Ala Val Val Gly 49 Glu Thr Asn His Ala Val Val Gly 52Glu Thr Lys Pro Ala Val Val Gly 53 Glu Thr His Pro Ala Val Val Gly 54Glu His Asn Pro Ala Val Val Gly 55 His Thr Asn Pro Ala Val Val Gly 56Pro Thr His Pro Ala Val Val Gly 57 Pro Thr Asn Pro Ala Val Val Gly 58His Thr His Pro Ala Val Val Gly 59 Glu Thr Phe Pro Ala Val Val Gly 60His Thr Phe Pro Ala Val Val Gly 61 Gly Thr Phe Pro Ala Val Val Gly 62His Thr Gly Pro Ala Val Val Gly 63 His Thr Lys Pro Ala Val Val Gly

For Table 4 above, the first column listing of the “Pro-Peptide SequenceName” corresponds to the SEQ ID NO for the indicated elastase cleavagedomain. Thus, 24 corresponds to the wild-type trypsin cleavage domain ofSEQ ID NO:24. Numbers 40-49, 52-63 correspond respectively to thevariant elastase cleavage domains of SEQ ID NOS: 40-49, and 52-63.

To culture the variant clones, shaker flask culture conditions developedfor the trypsin-activated 201-24-266-VU clone described in the precedingExample were generally followed. Several methods of converting thevariant proproteins secreted into the shaker flask supernatant to maturePRT-201 were tested. The first conversion strategy consisted ofchromatographically purifying the variant proenzyme first, followed bycontrolled cleavage in a specific conversion buffer. Because the aminoacid changes in the variant proproteins resulted in only small changesin the theoretical isoelectric points compared to the wild-typeproenzyme, cation exchange chromatography was carried out generally asdescribed in the preceding Example. Supernatants from the variant clonecultures were prepared for chromatography either by dilution with watergenerally as described in the preceding Example or by concentrationfollowed by diafiltration of the supernatant using tangential flowfiltration into the column loading buffer. After chromatographicpurification, eluted fractions were analyzed by SDS-PAGE followed byCoomassie staining. Gel analysis demonstrated greater amounts ofconverted mature protein to proprotein in the fractions compared to thestarting supernatant, indicating that a considerable amount ofspontaneous proprotein conversion had occurred. Upon subsequentpurification, the spontaneously converted protein was determined byLC/MS to consist of mainly N-terminal variants which were shown to havelittle or no elastase activity in the SLAP assay.

The second conversion strategy consisted of converting the variantproprotein prior to purifying from the culture supernatant, followed bychromatographic purification of the mature enzyme. This conversionstrategy was first tested in a small-scale assay and subsequently scaledup to accommodate larger conversion volumes. For small-scale conversion,clarified supernatant from variant clone cultures was typicallyconcentrated 5-fold by centrifugation at 2-8° C. in an ultracentrifugalfilter device. After concentration, the retentate was diluted 5-foldwith Tris buffer to a final concentration of 100 mM of Tris-HCltypically in a pH range of 8.0 to 9.0. Samples were incubated at roomtemperature on a rocking platform. Elastase activity was monitored bySLAP assay typically until the activity reaction velocity reached aplateau. In some cases, the reaction velocity increased so slowly thatSLAP monitoring was halted before a plateau was achieved. Convertedsamples were analyzed for protein species (e.g., proprotein andPRT-201/N-terminal variants) by SDS-PAGE. To scale up this conversionstrategy, supernatant containing variant proprotein was concentrated10-fold using tangential flow filtration followed by diafiltration with100 mM Tris-HCl ranging in pH from 6.0 to 9.0. The progress of theconversion reaction was monitored over time by HIC-HPLC analysis inwhich proprotein, PRT-201, and N-terminal variant species werequantified. When the pH of the diafiltration buffer was between 8.0 and9.0 there were generally higher rates of conversion.

The elastase proproteins listed in Table 5 were expressed in P. pastorisas described and tested for their capacity to undergo auto-conversion insmall-scale conversion assays as described in the second conversionstrategy above. The results of those studies are summarized in Table 5below:

TABLE 5 Results of expression of elastase proproteins in P. pastoris.Pro-Peptide Shaker Flask Shaker Flask % N-Terminal Trypsin Used inSequence Name Yield Stability Conversion Rate Variants Processing 24High High Fast 20% Yes 40 None Not Applicable Not Applicable NotApplicable No 41 Intermediate High Slow Not Tested No 42 Low LowIntermediate 25% No 43 Intermediate High Slow Not Tested No 44Intermediate-High High No Conversion Not Applicable No Detected 45Intermediate High No Conversion Not Applicable No Detected 46Intermediate High No Conversion Not Applicable No Detected 47Intermediate High No Conversion Not Applicable No Detected 48Intermediate Low Fast 15% No 49 High High Slow 35% No 52 IntermediateLow Fast Not Tested No 53 Intermediate Intermediate Fast 25% No 54Intermediate Low Fast Not Tested No 55 Intermediate - Intermediate Fast15% No High 56 Low Low Not Tested Not Tested No 57 Low Low Not TestedNot Tested No 58 Intermediate - Intermediate Slow Not Tested No High 59Intermediate - Intermediate Slow Not Tested No High 60 Intermediate -High Slow Not Tested No High 61 None Not Applicable Not Applicable NotApplicable No 62 High High No Conversion Not Applicable No Detected 63None Not Applicable Not Applicable Not Applicable No

In Table 5 above, the first column listing of the “Pro-Peptide SequenceName” corresponds to the SEQ ID NO for the indicated elastase cleavagedomain. Thus, 24 corresponds to the wild-type trypsin activated elastasecleavage domain of SEQ ID NO:24. Numbers 40-49, and 52-63 correspondrespectively to the variant elastase cleavage domains of SEQ ID NOS:40-49, and 52-63. The column labeled “Shaker Flask Yield” corresponds tothe amount of the corresponding proprotein in the culture supernatantover 3 days of induction as determined by SDS-PAGE analysis. The columnlabeled “Shaker Flask Stability” corresponds to the stability of thecorresponding proprotein in the supernatant of the shaker flask culturemedia over 3 days of induction as determined by the amount ofPRT-201/N-terminal variants seen on SDS-PAGE analysis. The columnlabeled “Conversion Rate” corresponds to the relative rate of conversionof proprotein to PRT-201, as indicated by the time to achieve maximalSLAP reaction velocity (Fast: less than 60 minutes; Intermediate: 60 to120 minutes; and Slow: greater than 120 minutes). Conversion timecourses of the variant proproteins were determined using the small-scaleconversion assay described above and compared to the conversion timecourse of the 24 proprotein determined by activation using immobilizedtrypsin. The column labeled “% N-Terminal Variants” refers to thepercentage of converted protein that comprised N-terminal variants ofthe mature elastase protein (i.e. variants comprising cleavage at thebond C-terminal to any site other than P1). To illustrate the relativeranking systems used in Table 5, examples of SDS-PAGE, conversion rateand N-terminal variant analyses for a subset of auto-activated variantsare shown in FIG. 5.

Analysis of the various variants revealed that elastase proproteinscomprising either the SEQ ID NO:48 or SEQ ID NO:55 variant elastasecleavage domain provided auto-activated elastases with superiorqualities including intermediate to high shaker flask yields and lowpercentages of variants upon conversion. Further analysis of elastaseproproteins comprising either the SEQ ID NO:48 and SEQ ID NO:55 variantelastase cleavage domain revealed that auto-activation of thecorresponding proproteins (i.e. the elastase proenzymes of SEQ ID NO:64and SEQ ID NO:69, respectively) produced just one class of N-terminalvariant with a cleavage at the peptide bond C-terminal to the P′2residue. Further analysis of elastase proproteins revealed that theproprotein comprising SEQ ID NO:55 variant elastase cleavage domain wasmore stablethan the proprotein comprising the SEQ ID NO:48 variantelastase cleavage domain.

Initial experiments to optimize conditions for controlled cleavage ofpurified pro-PRT-201 were performed using the proprotein with the 42pro-peptide sequence (SEQ ID NO:6). This purified proprotein wassubjected to conversion in a matrix of conditions including pH (7.7 to8.9), buffer composition (0.4 to 10 mM sodium citrate), proteinconcentration (0.14 to 0.23 mg/mL), and reaction time (5 to 24 hours).At the end of the conversion period, the reactions were quenched byadding formic acid to reduce the pH to 3.0. The relative amounts ofprotein species in each reaction were determined by mass spectrometry.Based on these results, the conversion conditions that resulted in thelowest percentage of N-terminal variants included a pH of 8.3, a buffercomposition of 100 mM Tris and less than 1 mM sodium citrate, a proteinconcentration of 0.2 mg/mL, and a reaction time of 5 to 24 hours. Inthis study, only the reaction end points were analyzed. Thus real-timedata on conversion quality was not obtained, and the final result mayhave reflected both the initial production of N-terminal variants and asubstantial amount of time for those variants to have been degraded.Subsequently, an HIC-HPLC assay was developed to enable real-timemonitoring of the conversion reaction. Further conversion optimizationstudies, including those using real-time HIC-HPLC monitoring, aredescribed in Example 6.

6.7 Example 6 Expression of Auto-Activated PRT-201 in P. pastoris Usinga Multicopy Variant Vector

Multicopy integration of recombinant genes in P. pastoris has beenutilized to increase expression of the desired protein (see, e.g.,Sreekrishna et al., 1989, Biochemistry 28:4117-4125; Clare et al., 1991,Bio/Technology 9:455-460; Romanos et al., 1991, Vaccine 9:901-906).However, in certain instances, expression levels obtained from singlecopy vector integrants was efficient and was not improved by themulticopy vector integrants (Cregg et al., 1987, Bio/Technology5:479-485). Spontaneous multicopy plasmid integration events occur invivo at a low frequency in P. pastoris. To obtain genomic integration ofmultiple copies of a gene and possibly increase the protein expression,an in vitro ligation method can be used to produce tandem inserts of thegene in an expression vector, followed by P. pastoris transformation.

To obtain a multicopy integrant of the pPROT55-V variant, an in vitroligation method was used to construct a vector containing multiplecopies of the pPROT55-V that was subsequently used for P. pastoristransformation. To make the multicopy vector, the pPROT55-V vector wasdigested with BglII and BamHI to release the 2.3 kb expression cassetteencoding the pro-PRT-201 gene, the AOX1 promoter, and the AOX1transcription termination sequence. The expression cassette was thenligated with a preparation of the pPROT55-V vector that had beenlinearized with BamHI and treated with calf intestinal alkalinephosphatase (New England Biolabs, MA, USA) to prevent self-ligating. Theligation mixture was incubated overnight at 16° C. The E. coli TOP10strain (Invitrogen, CA, USA) was transformed with the ligation reaction.The transformation mix was plated out on low salt LB in the presence of25 microgram/mL of Zeocin. Drug-resistant transformants were picked andplasmid DNA was prepared.

To determine the number of expression cassettes in the resulting clones,plasmid DNA was digested with BglII and BamHI and analyzed by agarosegel electrophoresis with a DNA size standard marker. A clone containinga single defined insert band with the size consistent with three 2.3 kbexpression cassettes was identified and named pPROT55M3-V. Restrictionenzyme mapping was used to confirm the orientation of a linearhead-to-tail multimer formation for the pPROT55M3-V vector. FIG. 6depicts the pPROT55M3-V cloning scheme.

The wild-type P. pastoris strain NRRL Y-11430 was used fortransformation, which was carried out as described in Example 4 exceptthat the pPROT55M3-V vector was linearized with BglII instead of SacIprior to transformation. Drug-resistant transformants were cultured andscreened for expression of the pPROT55M3 proprotein as described inExample 4.

Optimization of shaker flask culture conditions was performed tominimize spontaneous cleavage during induction. Shaker flaskoptimization focused on two variables, induction temperature andinduction media composition. First, it was found that performinginduction at 22° C. compared to 25° C. resulted in a higher ratio ofproenzyme to mature enzyme in the culture supernatant for all mediacompositions tested. Second, addition of sodium citrate to increase thebuffering strength of the induction media resulted in the absence ofspontaneously converted mature enzyme in the culture supernatant acrossall sodium citrate concentrations tested (12.5 to 50 mM). The effects ofthese variables on proprotein expression yield and stability in shakerflask supernatant over time are illustrated in FIG. 7.

The high-expressing three-copy clone 201-55M3-003-VU was selected forscale-up fermentation analysis using fermentation procedures establishedfor the 201-55-001-VU clone described as follows. Fermentation of the201-55-001-VU clone was effected by thawing one cell bank vial and usingit to inoculate a shaker flask containing 500 ml of BKGY growth mediumat pH 5.7. The seed culture was grown for 24 hours with shaking at 28°C. until the wet cell weight was approximately 40 g/L. The fermentorcontaining BKGY growth medium at pH 5.7 was sterilized in the autoclave.After the media was cooled to 28° C., supplements including yeastnitrogen base and biotin were added. The fermentor was inoculated at aratio of 1:33 of seed culture to BKGY growth medium.

The fermentation procedure started with a fed-batch of glycerol andglycerol feed at pH 5.7 at 28° C. The pH was controlled by 10%phosphoric acid and 30% ammonium sulfate solutions. The culture wasagitated from 300-1000 rpm with aeration to control the dissolved oxygenat 40%. After the initial glycerol batch was depleted and dissolvedoxygen spiked, indicating the depletion of glycerol from the system,additional 50% glycerol was fed at 131 g/h until the wet cell weightreached preferably between 200 g/L to 300 g/L. After the wet cell weightreached 200 g/L-300 g/L, the induction was immediately initiated with amethanol bolus of 0.025 mL per gram of wet biomass. After depletion ofthe methanol bolus and the rise of dissolved oxygen, induction wascontinued by the addition of limiting amounts of methanol with aconstant feed rate of 0.0034 g methanol/g wet cell weight/hr. At thestart of constant methanol feed, the pH of the fermentation broth waschanged from 5.7 to 5.5 and the temperature was changed from 28° C. to22° C. The fermentation was harvested after 70 hours of induction.

To determine the relative yields of single and 3-copy clones, clone201-55-001-VU, containing one genomic integrant of the single copypPROT55-V vector, was fermented in parallel with clone 201-55M3-003-VU,containing one genomic integrant of the 3-copy pPROT55M3-V vector. Thefermentations were carried out as described above except that the pH ofthe culture was maintained at 5.7 throughout induction. The harvestedsupernatants from 201-55-001-VU and 201-55M3-003-VU fermentations wereanalyzed by gradient SDS-PAGE followed by Colloidal Blue staining (FIG.8), which showed that higher proprotein expression was obtained from themulticopy 201-55M3-003-VU clone compared to the single copy201-55-001-VU clone. SDS-PAGE results were confirmed with HIC-HPLCanalysis of proprotein concentration in the fermentation supernatants,demonstrating that the 201-55M3-003-VU clone produced approximately 600mg/L of the secreted proprotein while the 201-55-001-VU clone producedapproximately 400 mg/L. Thus, the multicopy 201-55M3-003-VU clonecontaining three expression cassettes produced approximately 50% moreproprotein compared to single copy 201-55-001-VU clone containing asingle expression cassette.

Two conversion strategies were tested using the 201-55M3-003-VUsupernatant produced from the fermentation. These strategies generallyfollow the strategies described for other proprotein variants in Example5, except that they were performed on a larger scale. In the firststrategy, the proprotein was captured from the supernatant by cationexchange chromatography, followed by conversion to the mature proteinand polish chromatography. In the second strategy, the proprotein wasconverted to the mature protein prior to purification, followed bycapture using cation exchange chromatography, extended conversion toremove the N-terminal variants, and further polish chromatography. Bothstrategies also included an extended incubation step in a buffer at pH8.0 after conversion to effect the selective degradation of N-terminalvariants.

Using the first strategy, capture of the proprotein followed byconversion and PRT-201 polish purification were effected as follows. The201-55M3-003-VU supernatant was harvested from the fermentation cultureand frozen at −80° C. Approximately 7 L of frozen clarified supernatant(3.5 L from the 201-55M3-003-VU fermentation described above and 3.5 Lfrom 201-55M3-003-VU shaker flask cultures prepared generally asdescribed in Examples 4 and 5) was thawed and diluted 8-fold withdeionized water and 1 M sodium citrate, pH 4.3, at 2-8° C. to obtain afinal concentration of 25 mM sodium citrate. The pH of the solution wasadjusted to 4.7. The solution was loaded onto a 2.3 L bed Macroprep HighS cation exchange column at 76 cm/hr at 2-8° C. The column was washedwith 5 CVs of 25 mM sodium citrate, pH 4.7, followed by 5 CVs of 160 mMsodium chloride, 25 mM sodium citrate, pH 4.7. The proprotein was elutedwith 15 CVs of a linear gradient starting from 160 mM sodium chloride to500 mM sodium chloride in 25 mM sodium citrate, pH 4.7, at 87 ml/min (67cm/hr). The eluate was collected in fractions. Fractions were analyzedby SDS-PAGE for protein content (FIG. 9). A small amount ofspontaneously converted protein was observed by SDS-PAGE. Fractionscontaining the proprotein were pooled for further processing. The pooledmaterial was subjected to HIC-HPLC analysis, which showed that itconsisted of 92% proprotein and 8% mature PRT-201.

To initiate conversion, the pooled material was buffer exchanged usingtangential flow filtration into 100 mM sodium chloride, 20 mM Tris, pH4.0, using constant volume diafiltration at 10-12° C. Tangential flowfiltration was performed with regenerated cellulose membranes, atransmembrane pressure of 15 psi, a crossflow rate of 20 L/min and aflux of 800 mL/min. Three diavolumes of the buffer at 2-8° C. was addedat the same rate as the flux. Subsequently, three additional volumes ofbuffer at ambient temperature were added at the same rate as the flux toraise the temperature of the conversion solution to the target of 26° C.Tangential flow filtration was used to concentrate the conversionsolution to the target of 1.5 mg/mL using the conditions of 15 psitransmembrane pressure, 1.2 L/min crossflow rate, and 76 ml/min flux.However, after approximately 2 minutes of starting the concentrationprocedure, unexpected precipitation was observed and the concentrationprocess was halted. The protein concentration of the conversion solutionwas determined to be 1.1 mg/mL by UV absorbance at 280 nm in a volume of570 mL. To minimize further precipitation, the conversion solution wasdiluted to 1 mg/mL with 100 mM sodium chloride, 20 mM Tris, pH 4.0, andfiltered through a 0.22 micron membrane. Sixteen mL of 3 M Tris, pH 9.0was added to the conversion solution. The conversion solution was placedin a water bath at 26° C. The conversion reaction was monitored byHIC-HPLC analysis. After 30 minutes, HIC-HPLC showed that the majorityof the proprotein had been converted to PRT-201 and some N-terminalvariants (FIG. 10). After 1 hour, the conversion reaction consisted of0% proprotein, 86% full-length PRT-201 and 14% N-terminal variants. Theconversion material was incubated further for 4 more hours, at whichtime HIC-HPLC analysis showed that the conversion material consisted of98% full-length PRT-201 and 2% N-terminal variants.

The conversion material was diluted 4-fold with deionized water and 1 Msodium citrate, pH 4.3, to a final concentration of 25 mM sodiumcitrate. The pH of the solution was adjusted to 5.0 in preparation forloading onto the polish column. The solution was loaded onto a 600 mLbed Macroprep High S cation exchange column at 27 mL/min (83 cm/hr). Thecolumn was washed with 5 CVs of 20 mM sodium citrate, pH 5.0 followed by5 CVs of 160 mM sodium chloride, 20 mM sodium citrate, pH 5.0. PRT-201was eluted with 15 CVs of a linear gradient starting from 160 mM to 500mM sodium chloride in 25 mM sodium citrate, pH 5.0, at 87 mL/min (67cm/hr). The eluate was collected in fractions. Fractions were analyzedby SDS-PAGE for protein content and by SLAP assay for specific activity.Fractions containing PRT-201 with a specific activity of ≧30 U/mg werepooled. The pooled PRT-201 fractions were diafiltered by tangential flowfiltration into formulation buffer (0.1×PBS, pH 5.0). The pH ofdiafiltered PRT-201 was adjusted to pH 7.4 and the protein concentrationwas adjusted to 1 mg/mL by tangential flow filtration. Vials were filledand lyophilized as described for PRT-201 produced from the 201-24-266-VUclone in Example 4.

Using the second strategy, proprotein conversion followed bypurification of PRT-201 was effected as follows. Approximately 7 L ofthe frozen clarified supernatant from the 201-55M3-003-VU fermentationdescribed above was thawed and the conversion reaction was initiated bytangential flow filtration with a conversion buffer containing 100 mMTris, pH 8.0, using constant volume diafiltration at ambient temperaturewith regenerated cellulose membranes. Two diavolumes of 100 mM Tris-HCl,pH 8.0 were sufficient to change the pH of the retentate from pH 5.0 to8.0 and effect conversion. An experiment was also performed by directlyadjusting the pH of the clarified supernatant to 8.0 by adding a Trisbase to a final concentration of 100 mM and adjusting the pH to 8.0 with1 N sodium hydroxide. This resulted in the formation of precipitates anda cloudy supernatant, possibly due to precipitation of broth components,which was undesirable. The preferred method of conversion usingtangential flow filtration did not result in precipitation.

The conversion reaction was monitored by real-time HIC-HPLC analysis.After initiation of conversion, pro-PRT-201 converted to mature PRT-201slowly over the first two hours, followed by an acceleration ofconversion (FIG. 11). At 4.5 hours, the conversion reaction consisted ofapproximately 4% pro-PRT-201, 75% mature PRT-201 and 21% N-terminalvariants. At 6.5 hours, the conversion reaction consisted ofapproximately 1% pro-PRT-201, 83% mature PRT-201 and 16% N-terminalvariants. The reduction in N-terminal variants from 21% to 16% from 4.5hrs to 6.5 hrs may be due to degradation of N-terminal variants by fulllength, active PRT-201, but this was not complete and not as rapid asthe reduction in N-terminal variants seen during conversion of purifiedpro-PRT 201. In this second strategy, extending the conversion reactionmay not entirely remove the N-terminal variants due to the competingproteins in the supernatant that compete for the active site ofelastase. To improve the conversion reaction, the postconversionmaterial was captured and subsequently diafiltered into an appropriatebuffer to initiate extended conversion at pH 8.0 for selectivedegradation of N-terminal variants as described below.

Capture of PRT-201 from the conversion material was effected as follows.The conversion material was buffer exchanged into 20 mM sodium citrate,pH 5.0 and loaded onto a Macro-Prep High S cation-exchangechromatography column. The column was washed with 5 CVs of 20 mM sodiumcitrate, pH 5.0 followed by 5 CVs of 20 mM sodium citrate, 160 mM sodiumchloride, pH 5.0. PRT-201 was eluted with a linear gradient from 160 mMto 500 mM sodium chloride in 20 mM sodium citrate, pH 5.0. The fractionswere analyzed by SDS-PAGE for protein content, HIC-HPLC for N-terminalvariants, UV absorbance at 280 nm for protein concentration, and SLAPassay for elastase activity. Two predominant proteins bands weredetected by SDS-PAGE, corresponding to PRT-201 and PRT-201 glycoforms,as shown in FIG. 12. Fractions that contained PRT-201 glycoforms asdetermined by SDS-PAGE or N-terminal variants as determined by HIC-HPLCwere excluded from pooling. Fractions that exhibited relatively lowspecific activity (less than 30 U/mg) as determined by SLAP assay werealso excluded from pooling. The remaining fractions containing PRT-201were pooled for further processing. HIC-HPLC analysis of the pooledfractions revealed that this material consisted of approximately 98%full-length PRT-201 and 1-2% N-terminal variants. The pooled materialwas stored at 2-8° C. for 12-16 hrs.

Given the prior observation that N-terminal variants appeared todecrease over a prolonged conversion period as described above, thepooled PRT-201 material was subjected to an extended incubation step atpH 8.0. The extended incubation was performed by diafiltration andtangential flow filtration with 100 mM Tris, 300 mM sodium chloride, pH8.0 for 2.5 hours at ambient temperature. After 2.5 hours, theconversion material consisted of 100% mature PRT-201 as shown byHIC-HPLC analysis. The conversion material was subjected todiafiltration and tangential flow filtration into 20 mM sodium citrate,pH 5.0 to suppress elastase activity and prepare for columnchromatography. The diafiltered PRT-201 material was stored forapproximately 64 hours at 2-8° C.

Polish chromatographic purification of PRT-201 was effected as follows.The diafiltered PRT-201 material was loaded onto a Macro-Prep High Scation exchange column and washed with 5 CVs of Buffer C (20 mM sodiumcitrate, pH 5.0) followed by 5 CVs of 160 mM sodium chloride, 20 mMsodium citrate, pH 5.0. Elution of PRT-201 was performed with a lineargradient of 15 CVs starting from 68% Buffer C and 32% Buffer D (160 mMsodium chloride, 25 mM sodium citrate, pH 5.0) to 0% Buffer C and 100%Buffer D (500 mM sodium chloride, 25 mM sodium citrate, pH 5.0), at 50ml/min (153 cm/hr). The eluate was collected in fractions. PRT-201eluted as a symmetrical peak at 37 mS/cm (330 mM sodium chloride).Fractions were analyzed by SDS-PAGE analysis for protein content, UVabsorbance at 280 nm for protein concentration and SLAP assay forspecific activity. Fractions containing PRT-201 that had a specificactivity of 30.1-38.8 U/mg were pooled. The pooled PRT-201 fractionswere diafiltered by tangential flow filtration into formulation buffer(0.1×PBS, pH 5.0). The pH of diafiltered PRT-201 was adjusted to pH 7.4and the protein concentration was adjusted to 1 mg/mL by tangential flowfiltration. Vials were filled and lyophilized as described for PRT-201produced from the 201-24-266-VU clone in Example 4.

The conditions for the conversion procedures described above were chosenbased on conversion optimization studies that examined proteinconcentration, temperature, buffer composition, diavolume and pHvariables. In the first study, the effect of proprotein concentration onthe production of N-terminal variants during conversion was analyzed.Purified pro-PRT-201 from the 201-55M3-003-VU clone(pro-PRT-201-55M3-003-VU) at a starting concentration of 0.2 mg/mL in 20mM sodium phosphate, pH 5.0 was aliquotted and concentrated to 1.0, 1.6,and 1.8 mg/mL using centrifugal concentrating devices as determined byUV absorbance at 280 nm. Conversion of the proprotein was effected byadding Tris and sodium chloride to 100 mM each of the four concentrationsamples, adjusting the pH from 5.0 to 8.0, and incubating the samples atambient temperature. The conversion reaction was monitored by HIC-HPLCin real-time until the proprotein was ≦1% of the total protein (FIG.13). At this endpoint, the 0.2 mg/mL sample consisted of approximately8% N-terminal variants, whereas the 1.0 mg/mL, 1.6 and 2.0 mg/mL samplesconsisted of approximately 14%, 19% and 29% N-terminal variants,respectively. The remainder of the protein in each sample consisted offull-length PRT-201. These results demonstrate that increasingconcentrations of pro-PRT-201-55-003-VU during conversion leads to theformation of more N-terminal variants and less full-length PRT-201.Other studies have suggested that pro-PRT-201-55M3-003-VU conversionoccurs through both intramolecular and intermolecular reactions. Thus,for this variant proprotein, it is likely that intramolecular reactions,which are favored in more dilute proprotein solutions, give rise to moreaccurate conversion whereas intermolecular reactions, favored in moreconcentrated proprotein solutions, result in less accurate conversion,i.e., the formation of a higher percentage of the N-terminal variantsrelative to full-length PRT-201.

In the second study, the effect of temperature on the production ofN-terminal variants during conversion was analyzed. Purified pro-PRT-201from the 201-55M3-VU clone (named pro-PRT-201-55M3-003-VU) was producedat a concentration of 1.6 mg/mL was subjected to conversion as describedabove at either 15° C. or 26° C. The conversion reactions were monitoredin real-time by HIC-HPLC and allowed to progress until the proproteincomprised <1% of total protein. The time required to reach thisreduction in proprotein was approximately 30 minutes at 26° C. andapproximately 90 minutes at 15° C. At these times, a similar percentageof N-terminal variants (about 20% of total protein) for bothtemperatures was observed. Thus, the higher temperature of 26° C.resulted in a more rapid conversion reaction compared to the lowertemperature of 15° C. while producing an essentially identical reactionproduct profile.

The third study examined the effect of buffer composition on proproteinsolubility during the conversion reaction. Purified pro-PRT-201(pro-PRT-201-55M3-003-VU) at a concentration of 1.0 mg/mL was subjectedto conversion under the conditions listed in Table 6. The conversionreactions were performed at ambient temperature except for one (buffercomposition of 20 mM Tris-HCl, 100 mM sodium chloride, pH 4.0) that wasperformed at 2 to 8° C. Conversion reactions were inspected visually forprecipitation. As noted in Table 6, buffer compositions that did notresult in precipitation included 100 mM Tris-HCl, pH 8.0; 100 mMTris-HCl, 100 mM sodium chloride, pH 5.0; and 100 mM Tris-HCl, 300 mMsodium chloride, pH 8.0. Buffer compositions with lower concentrationsof Tris or without sodium chloride at lower pH (i.e., pH 5.0) exhibitedprecipitation.

TABLE 6 Effect of buffer composition on precipitation during conversion.Soluble (No Precipitation Precipitation Buffer Composition TemperatureObserved Observed) 1 mM Tris-HCl, pH 5.0 Ambient + 25 mM Tris-HCl, pH5.0 Ambient + 100 mM Tris-HCl, pH 5.0 Ambient + 100 mM Tris-HCl, pH 8.0Ambient + 20 mM Tris-HCl, 100 mM 2-8° C. + sodium chloride, pH 4.0 100mM Tris-HCl, 100 mM Ambient + sodium chloride, pH 5.0 100 mM Tris-HCl,300 mM Ambient + sodium chloride, pH 8.0

In the fourth study, the effect of tangential flow filtration diavolumenumber on precipitation during conversion of supernatant containingproprotein (pro-PRT-201-55M3-003-VU) was analyzed. The solution used forbuffer exchange was 100 mM Tris-HCl, 100 mM sodium chloride, pH 8.0.Additionally, a direct pH adjustment of the supernatant from pH 5.0 to8.0 without tangential flow filtration was tested. As noted in Table 7,direct pH adjustment of the supernatant resulted in a large amount ofprecipitation. With 1 diavolume of exchange, some precipitation wasobserved. No precipitation was observed when 2 to 5 diavolumes wereused.

TABLE 7 Effect of diavolume number on precipitation during bufferexchange. Diavolumes Precipitation Observed 0 Major precipitation 1Minor precipitation 2 No precipitation 3 No precipitation 5 Noprecipitation

In the fifth study, the effect of pH on elastase activity of maturePRT-201 was analyzed. This study was designed to identify a useful pHrange for conversion that would not result in an irreversible loss ofelastase activity of the mature PRT-201 conversion product. Solutions of1 mg/mL PRT-201 in 20 mM Tris-HCl, 20 mM potassium phosphate wereprepared from pH 1 to 14. Solutions were kept at ambient temperature for0.5, 2, 24 and 48 hours. At the indicated time points, the solutionswere tested for elastase activity in the SLAP assay. Elastase activitywas largely stable from pH 3 to 8 at all time points. At pHs less than 3and greater 8, elastase activity was reduced at all time points, with acorrelation between longer time points and lower elastase activity.These results indicated that a conversion reaction performed outside apH range of 3 to 8 could negatively impact elastase activity of thePRT-201 conversion product.

6.8 Example 7 Production of Auto-Activated Recombinant Porcine Type IPancreatic Elastase

A vector encoding auto-activated porcine ELA-1 proenzyme was expressedP. pastoris. The resulting auto-activated porcine ELA-1 was compared toa porcine ELA-1 protein expressed as a trypsin-activated wild-typeproprotein.

To construct the trypsin-activated porcine ELA-1 vector, the porcineELA-1 coding region was synthesized by Blue Heron Biotechnology(Bothell, Wash.) using a non-PCR “long oligo” technique under licensefrom Amgen (Thousand Oaks, Calif.). The recombinant gene was cloned intothe Blue Heron pUC vector, a derivative of pUC119. The porcine ELA-1gene was sequenced on both strands to confirm the correct sequence.SacII and Xbal restriction sites were incorporated as potential cloningsites flanking the porcine ELA-1 gene as shown in FIG. 14. A second stopcodon was also added immediately after the native stop codon to minimizepotential ribosome read through. FIG. 15 shows the nature identicalamino acid sequence of porcine ELA-1 proenzyme, which contains thetrypsin-activated site.

The coding region of porcine ELA-1 was amplified by PCR using a pair ofoligonucleotides containing XhoI and SacII restriction sites tofacilitate cloning. The PCR product was digested with XhoI and SacII andpurified by agarose gel electrophoresis. The porcine ELA-1 fragment wascloned into the PV-1 vector at XhoI and SacII restriction sites. The E.coli TOP10 strain was transformed with the ligation mixture. The cellmixture was plated on low salt LB plates supplemented with 25 mg/mLZeocin. Drug-resistant clones were picked and plasmid DNA was prepared(Qiagen, CA). Based on restriction analysis, a clone containing theporcine ELA-1 gene insert was identified and the vector was namedpPROT101-24-V. The coding sequence of this vector was confirmed by DNAsequencing. The cloning scheme for pPROT101-24-V is depicted in FIG. 16.

Using the trypsin-activated pPROT101-24-V vector, three differentauto-activated clones were engineered by changing the trypsin cleavagesite in the pro-peptide region of porcine ELA-1 to elastase cleavagesites. Site-directed mutagenesis was performed generally as described inExample 4 using synthetic oligonucleotide primers containing the desiredmutations as described in Table 8. All the mutations in the pro-peptideregion were confirmed by double-stranded DNA sequencing.

TABLE 8 Cleavage domain sequences of trypsin-activated andauto-activated porcine ELA-1 vectors. Mutagenized codons are shaded. Thecleaved bond is between P1 and P′1. SEQ Pro-peptide sequence Maturesequence ID Construct name P7 P6 P5 P4 P3 P2 P1 P′1 P′2 P′3 NO.Trypsin-activated Phe Pro Glu Thr Asn Ala Arg Val Val Gly 115pPROT101-24-V Auto-activated Phe Pro Glu Thr Asn Ala Ala Val Val Gly 116pPROT101-42-V Auto-activated Phe Pro Glu Thr Asn His Ala Val Val Gly 118pPROT101-49-V Auto-activated Leu Pro His Thr Asn Pro Ala Val Val Gly 117pPROT101-55L-V

The wild-type NRRL Y-11430 P. pastoris strain was transformed anddrug-resistant transformants were cultured and screened for expressionof the porcine ELA-1 proproteins as described in Example 3. Based onanalysis by SDS-PAGE and Coomassie staining (see FIG. 17 and FIG. 18),the wild-type trypsin-activated pPROT101-24-V clones had the highestlevels of expression compared to the auto-activated clones. Of theauto-activated clones, the pPROT101-49-V clones had the highest level ofexpression, followed by the pPROT101-55L-V clones and then thepPROT101-49-V clones. Auto-activated pPROT101-42-V and pPROT101-55L-Vproproteins exhibited substantial spontaneous conversion duringinduction, while the trypsin-activated pPROT101-24-V and auto-activatedpPROT101-49-V proproteins showed greater stability in the inductionmedia.

Studies of the elastase activity of PRT-102 produced by trypsinactivation of proelastase protein expressed from pPROT101-24-V showedhigher specific activity than PRT-201 as shown in Table 9 below:

TABLE 9 Elastase activity of three different samples of mature porcinetype I pancreatic elastase (trypsin activated) as compared to maturehuman type I pancreatic elastase. Sample Name PRT-201 PRT-102 PRT-102PRT-102 Activity as measured by 34.6 91.8 99.4 100.7 SLAP (U/mg protein)32.9 88.3 91.3 88.6 (Replicates) Average of Replicates 33.6 88.5 93.592.9 Standard Deviation 0.9 3.2 5.2 6.7

A small-scale conversion experiment was used to determine ifpPROT101-55L-V and pPROT101-49-V proproteins could be converted tomature enzymes exhibiting elastase activity. pPROT101-55L-V andpPROT101-49-V shaker flask supernatants were concentrated 10-fold withan centrifugal filter unit and diluted 5-fold with 100 mM Tris-HCl, pH9.0. The conversion was allowed to proceed at room temperature andelastase activity was monitored over time by SLAP assay. The averagechange in absorbance per minute was determined from each time point andreported as non-normalized reaction velocity (FIG. 19). Conversion ofboth pPROT101-49-V and pPROT101-55L-V supernatants resulted in anincrease in elastase activity. The final time point samples from eachclone were analyzed by SDS-PAGE followed by Coomassie staining andcompared to pre-conversion samples (FIG. 20). The SDS-PAGE resultsconfirmed that nearly all of the pPROT101-49-V proprotein was convertedto mature protein by the end of the conversion assay. The SDS-PAGEresults also showed that nearly all of the pPROT101-55L-V had beenspontaneously converted to mature protein prior to the conversion assay.

Purified preparations of pPROT101-24-V and pPROT101-49-V proproteins andmature enzymes were submitted to Danforth Plant Science Center, MO forintact molecular weight analysis. The proproteins were purified bycation exchange chromatography (Macroprep High S, Bio-Rad). For matureenzyme analysis, the proproteins from both clones were first treated toproduce mature enzymes and then purified by cation exchangechromatography. The trypsin-activated proprotein was treated withtrypsin while the auto-activated proprotein was converted in thepresence of 100 mM Tris-HCl, pH 9.0, followed by cation exchangechromatography. The major peaks obtained from mass spectrometry analysisare listed in Table 10.

TABLE 10 Expected and observed molecular weights for porcine ELA-1proteins. Expected Observed Protein MW MW Trypsin-activatedpPROT101-24-V proprotein 27068 27064 Trypsin-activated pPROT101-24-Vmature 25908 25898 enzyme Auto-activated pPROT101-49-V proprotein 2704927047 Auto-activated pPROT101-49-V mature enzyme 25908 25910

SDS-PAGE, elastase activity and mass spectrometry results demonstratedthat auto-activated forms of type I porcine pancreatic elastase can beproduced by engineering the pro-peptide sequence to replace the trypsincleavage site with an elastase cleavage site. The expression levels ofthese auto-activated forms of type I porcine pancreatic elastase arelower than the wild-type trypsin-activated form. Of the autoactivatedclones tested, those with the pPROT101-49-V pro-peptide sequence showedthe highest level of expression and the least spontaneous conversion.Conversion of the pPROT101-49-V and pPROT101-55L-V clones resulted inthe production of mature proteins with substantial elastase activity.Mass spectrometry analysis revealed that the molecular weights ofpPROT101-49-V proprotein and mature porcine type I corresponded to theexpected masses.

6.9 Example 8 Trypsin Activity Analysis of Mature Recombinant HumanElastase-1 by Benz Colorimetric Peptide Substrate Assay

A colorimetric hydrolysis assay using the small peptide substrateN-benzoyl-Phe-Val-Arg-pNitroanilide (BENZ) was performed to determine ifpurified mature elastase protein produced by the auto-activated clone201-55M3-003-VU possesses trypsin activity. Three vials of lyophilizedPRT-201 purified from clone 201-55M3-003-VU were retrieved from −80° C.storage and reconstituted with water to obtain 1 mg/mL PRT-201 in0.1×PBS, pH 7.4. Protein concentrations were confirmed by measuring UVabsorbance at 280 nm. A TrypZean stock solution (10 mg/mL) was used togenerate a standard curve for trypsin activity. A previously testedtrypsin-activated PRT-201 sample was included as a positive control. Inaddition, some experimental and control samples were spiked withTrypZean to determine trypsin activity recovery in the presence ofPRT-201. A subset of the spiked and unspiked samples was treated withsoybean trypsin inhibitor (SBTI) to determine the ability of SBTI toinhibit any intrinsic or spiked trypsin activity. TrypZean standardswere also treated with SBTI to confirm the effectiveness of theinhibitor. See Table 11 below for a summary of the samples included inthis study.

TABLE 11 TrypZean dilutions for the standard curve were prepared usingthe assay buffer (0.1M Tris, pH 8.3). The standard curve for TrypZeansolutions is shown in FIG. 21. Plus TrypZean Plus SBTI No spike (to 10Plus TrypZean Description addition (to 100 ng/mL) ug/mL) spike and SBTIPRT-201 from clone 55M3 Vial #1 ✓ ✓ ✓ ✓ PRT-201 from clone 55M3 Vial #2✓ ✓ ✓ ✓ PRT-201 from clone 55M3 Vial #3 ✓ ✓ ✓ ✓ PRT-201 from trypsinactivated clone ✓ ✓ ✓ ✓ Buffer only (0.1M Tris, pH 8.3) ✓ Not done ✓ Notdone TrypZean standard, 1.56 ng/mL ✓ Not done ✓ Not done TrypZeanstandard, 3.13 ng/mL ✓ Not done ✓ Not done TrypZean standard, 6.25 ng/mL✓ Not done ✓ Not done TrypZean standard, 12.5 ng/mL ✓ Not done ✓ Notdone Tr5ypZean standard, 25 ng/mL ✓ Not done ✓ Not done TrypZeanstandard, 50 ng/mL ✓ Not done ✓ Not done TrypZean standard, 100 ng/mL ✓Not done ✓ Not done

The substrate solution was prepared (0.4 mg/mLN-benzoyl-Phe-Val-Arg-pNitroanilide Lot 7733 in 0.1 M Tris, pH 8.3) andprewarmed to 30° C. in a water bath. In triplicate, 100 microliters ofeach sample was pipetted into a 96-well microplate. Using a multichannelpipettor, 200 microliters of substrate solution was pipetted into eachwell and the microplate was immediately placed into a microplate readerpreheated to 30° C. The microplate reader recorded the absorbance at 405nm for each well once per minute for 60 minutes.

The PRT-201 samples were also tested for elastase activity in the SLAPassay. The SLAP substrate solution was prepared (4.5 mg/mL SLAP in 0.1 MTris, pH 8.3) and prewarmed to 30° C. in a water bath. The 1 mg/mLPRT-201 samples were diluted 20× with water to 0.05 mg/mL. Intriplicate, 10 microliters of each sample dilution was pipetted into a96-well microplate. Using a multichannel pipettor, 300 microliters ofSLAP substrate solution was pipetted into each well and the microplatewas immediately placed into a microplate reader preheated to 30° C. Themicroplate reader recorded the absorbance at 405 nm for each well onceper minute for 5 minutes.

The results of the BENZ and SLAP activity assays are respectivelypresented in Tables 11 and 12 below.

TABLE 12 Mean trypsin activity, reported as TrypZean concentrationequivalent (ng/mL). Plus TrypZean Plus SBTI spike (to 10 Plus TrypZeanDescription No addition (to 100 ng/mL) ug/mL) spike and SBTI PRT-201from clone 55M3 Vial #1    <1.56 [a] 118.7 <1.56 [a] <1.56 [a] PRT-201from clone 55M3 Vial #2    <1.56 [a] 122.4 <1.56 [a] <1.56 [a] PRT-201from clone 55M3 Vial #3    <1.56 [a] 122.8 <1.56 [a] <1.56 [a] PRT-201from trypsin activated clone 8.7 130.0 <1.56 [a] <1.56 [a] Buffer only(0.1M Tris, pH 8.3) 1.3 Not done <1.56 [a] Not done TrypZean standard,1.56 ng/mL 2.7 Not done <1.56 [a] Not done TrypZean standard, 3.13 ng/mL3.7 Not done <1.56 [a] Not done TrypZean standard, 6.25 ng/mL 6.5 Notdone <1.56 [a] Not done TrypZean standard, 12.5 ng/mL 11.2  Not done<1.56 [a] Not done Tr5ypZean standard, 25 ng/mL 23.5  Not done <1.56 [a]Not done TrypZean standard, 50 ng/mL 49.4  Not done <1.56 [a] Not doneTrypZean standard, 100 ng/mL 102.4  Not done <1.56 [a] Not done [a]Coefficient of regression < 0.8.

TABLE 13 Mean SLAP activity, reported as U/mg Description No additionPRT-201 from clone 55M3 Vial #1 34.9 PRT-201 from clone 55M3 Vial #236.6 PRT-201 from clone 55M3 Vial #3 35.7 PRT-201 from trypsin activatedclone 32.1

The level of trypsin activity of PRT-201 from clone 201-55M3-003-VU wasbelow the range of the standard curve in the trypsin activity assay(<1.56 ng/mL). Additionally, the coefficients of regression for thetriplicate hydrolysis reactions were poor (<0.8), further supporting theabsence of trypsin activity in this auto-activated mature elastaseprotein. In contrast, the level of trypsin activity of the controltrypsin-activate sample was determined to be 8.7 ng/mL.

6.10 Example 9 Drug Product Formulation

A study was conducted to identify lyophilized PRT-201 formulations thathad the following characteristics: strong ionic strength buffer uponreconstitution; pH stability at 7.4; capable of one-step reconstitution;improved stability; and improved cake appearance. A screen conducted toassess various pre-formulation buffers and excipients. Formulations withsodium phosphate buffer and trehalose and mannitol as excipients werefound to meet the study objectives. One specific lyophilized formulationhaving these characteristics produces upon reconstitution with water a 4mg/mL pH 7.4 solution of PRT-201 in 25 mM sodium phosphate, 3% mannitol,and 1% trehalose.

Thus, in certain embodiments, a formulation of the disclosure containssodium phosphate buffer and/or trehalose and/or mannitol. Suchformulations can be modified, e.g., by the addition of sodium chloride,so as to produce a more isotonic solution upon reconstitution.Alternatively, the formulation can be modified following reconstitutionso as to produce a more isotonic solution. In specific embodiments, theformulation is a unit dosage. In other embodiments, the formulation hasa molarity ranging from 120 to 150 mmol/L, more preferably ranging from135 to 140 mmol/L (for example 138 mmol/L).

SEQUENCE LISTING SEQ ID NO. Description Type of sequence Sequence   1Mature human Amino acid, single VVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTLelastase I, letter format,  IRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGTincluding first wherein: EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV “valine”X = V or L TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN   2 Mature human Amino acid, singleVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTLI elastase I, letter format, RQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT minus first wherein:EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV “valine” X = V or LTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV TSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN  3 Mature human Amino acid, single GGTEAGRNSWPSQISLQYRSGGSRYHTCGGTLIRelastase I, letter format,  QNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGTEminus first two wherein: QYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSVT “valines”X = V or L LNSYVQLGVLPQEGAILANNSPCYITGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMV CAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN   4 Mature human Amino acid, singleAVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTL elastase I, letter format, IRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT with first wherein:EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV “valine” X = V or LTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN substituted byGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM “alanine”VCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV TSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN  5 Mature human Amino acid, single VVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTLelastase I letter format IRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT (isotype 2),EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV including firstTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN “valine”GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM VCAGGDGSSLWMPG   6 EngineeredAmino acid, single TQDLPETNAAVVGGTEAGRNSWPSQISLQYRSGG elastaseletter format,  SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no.wherein DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 1 (pPROT42 X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI variant)TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN   7 EngineeredAmino acid, single TQDLPETNAAAVGGTEAGRNSWPSQISLQYRSGG elastaseletter format,  SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no.wherein: DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 2 X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN   8 Engineered Amino acid, singleTQDLPETAAAVVGGTEAGRNSWPSQISLQYRSGG elastase letter format, SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no. wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 3 X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN   9 Engineered Amino acid, singleTQDLPETNNAPVGGTEAGRNSWPSQISLQYRSGG elastase letter format, SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no. wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 4 X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV NGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN  10 Wild-type Amino acid, singleTQDLPETNARVVGGTEAGRNSWPSQISLQYRSGG elastase letter format, SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG proprotein no. wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY 5 (produced X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI fromTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY pPROT24WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV trypsinNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS activated WINNVIASN sequence)  11Consensus Amino acid, three Xaa₁ Xaa₂ Xaa₃ elastase letter format Xaa₁ =alanine, leucine, isoleucine,  recognitionmethionine, lysine, asparagine or valine sequence 1 Xaa₂ =proline, alanine, leucine,  (Positionsisoleucine, glycine, valine, or threonine Xaa₁ = P3, Xaa₃ =alanine, leucine, valine,  Xaa₂ = P2, isoleucine, or serine Xaa₃ = P1) 12 Consensus Amino acid, three Xaa₁ Pro Xaa₂ elastase letter formatXaa₁ = alanine, leucine, isoleucine,  recognitionmethionine, lysine, or valine sequence 2 Xaa₂ =alanine, leucine, valine,  (Positions P3- isoleucine, or serine P2-P1) 13 Consensus Amino acid, three Xaa₁ Xaa₂ Xaa₃ elastase letter formatXaa₁ = asparagine or alanine recognition Xaa₂ = proline or alaninesequence 3 Xaa₃ = alanine or leucine or valine (Positions P3- P2-P1)  14Elastase Amino acid, three Ala Ala Ala recognition letter formatsequence 1 (Positions P3- P2-P1)  15 Elastase Amino acid, threeAsn Ala Ala recognition letter format sequence 2 (Positions P3- P2-P1) 16 Elastase Amino acid, three Asn Ala Pro recognition letter formatsequence 3 (Positions P3- P2-P1)  17 Wild-type Amino acid, threeAsn Ala Arg trypsin letter format recognition sequence (pPROT24)(Positions P3- P2-P1)  18 Elastase Amino acid, three Ala Pro Alarecognition letter format sequence 5 (Positions P3- P2-P1)  19 ElastaseAmino acid, three Ala Ala Pro recognition letter format sequence 6(Positions P3- P2-P1)  20 Elastase Amino acid, three Asn Pro Alarecognition letter format sequence 7 (Positions P3- P2-P1 of Variants 48and 55)  21 Elastase Amino acid, three Leu Pro Ala recognitionletter format sequence 8  22 Human Amino acid, threeThr Gln Asp Leu Pro Glu Thr Asn Ala Arg elastase letter formatactivation sequence 1 (Wild-type)  23 Human Amino acid, threeThr Gln Asp Leu Pro Glu Thr Asn Ala Ala elastase letter formatactivation sequence 2 131 pro-PROT- Amino acid, threeThr Asn Ala Arg Val Val Gly Gly 201 cleavage letter format site  25pPROT40 Amino acid, three Thr Ala Ala Ala Val Val Gly Gly cleavage siteletter format  26 pPROT41 Amino acid, threeThr Asn Ala Ala Ala Val Gly Gly cleavage site letter format  27 pPROT42Amino acid, three Thr Asn Ala Ala Val Val Gly Gly cleavage siteletter format  28 pPROT43 Amino acid, threeThr Asn Ala Pro Val Val Gly Gly cleavage site letter format  29 pPROT44Amino acid, three Thr Gly Ala Gly Ile Val Gly Gly cleavage siteletter format  30 pPROT45 Amino acid, threeThr Val Pro Gly Val Val Gly Gly cleavage site letter format  31 pPROT46Amino acid, three Thr Ala Pro Gly Val Val Gly Gly cleavage siteletter format  32 pPROT47 Amino acid, threeThr Asn Pro Gly Val Val Gly Gly cleavage site letter format  33Coding region Nucleotide ACCCAGGACCTTCCGGAAACCAATGCCCGCGTA of a humanGTCGGAGGGACTGAGGCCGGGAGGAATTCCTG elastase-1GCCCTCTCAGATTTCCCTCCAGTACCGGTCTGG (NCBIAGGTTCCCGGTATCACACCTGTGGAGGGACCCT Accession No.TATCAGACAGAACTGGGTGATGACAGCTGCTCA NM_001971)CTGCGTGGATTACCAGAAGACTTTCCGCGTGGT GGCTGGAGACCATAACCTGAGCCAGAATGATGGCACTGAGCAGTACGTGAGTGTGCAGAAGATCGT GGTGCATCCATACTGGAACAGCGATAACGTGGCTGCCGGCTATGACATCGCCCTGCTGCGCCTGGC CCAGAGCGTTACCCTCAATAGCTATGTCCAGCTGGGTGTTCTGCCCCAGGAGGGAGCCATCCTGGCT AACAACAGTCCCTGCTACATCACAGGCTGGGGCAAGACCAAGACCAATGGGCAGCTGGCCCAGACC CTGCAGCAGGCTTACCTGCCCTCTGTGGACTACGCCATCTGCTCCAGCTCCTCCTACTGGGGCTCC ACTGTGAAGAACACCATGGTGTGTGCTGGTGGAGATGGAGTTCGCTCTGGATGCCAGGGTGACTCT GGGGGCCCCCTCCATTGCTTGGTGAATGGCAAGTATTCTGTCCATGGAGTGACCAGCTTTGTGTCCA GCCGGGGCTGTAATGTCTCCAGGAAGCCTACAGTCTTCACCCAGGTCTCTGCTTACATCTCCTGGAT AAATAATGTCATCGCCTCCAACTGA  34Yeast alpha Amino acid, threeMet-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- factor signalletter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr- peptide 35 20F primer Nucleotide Ggctcgagaaaagagaggctgaagctactcaggaccttccggaaaccaatgcccgg  36 24R primer Nucleotide gggccgcggcttatcagttggaggcgatgacat 37 pPROT42 P3 Amino acid, single AAVVGGTEAGRNSWPSQISLQYRSGGSRYHTCGcleavage site letter format,  GTLIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQN variantwherein: DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA elastase X = V or LQSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT KTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSX HGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN  38 pPROT42 P2 Amino acid, single AVVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTcleavage site letter format,  LIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDG variantwherein: TEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQS elastase X = V or LVTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKT NGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG VTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 39 Mature Amino acid, single VVGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTL porcineletter format IRQNWVMTAAHCVDRELTFRVVVGEHNLNQNDGT pancreaticEQYVGVQKIVVHPYWNTDDVAAGYDIALLRLAQSV elastase ITLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTN (fromGQLAQTLQQAYLPTVDYAICSSSSYWGSTVKNSM GenBankVCAGGDGVRSGCQGDSGGPLHCLVNGQYAVHGV AccessionTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN P00772.1)  40 ElastaseAmino acid, three Glu Thr Ala Ala Ala Val Val Gly variant letter formatpropeptide cleavage domain 40  41 Elastase Amino acid, threeGlu Thr Asn Ala Ala Ala Val Gly variant letter format propeptidecleavage domain 41  42 Elastase Amino acid, threeGlu Thr Asn Ala Ala Val Val Gly variant letter format propeptidecleavage domain 42  43 Elastase Amino acid, threeGlu Thr Asn Ala Pro Val Val Gly variant letter format propeptidecleavage domain 43  44 Elastase Amino acid, threeGlu Thr Gly Ala Gly Ile Val Gly variant letter format propeptidecleavage domain 44  45 Elastase Amino acid, threeGlu Thr Val Pro Gly Val Val Gly variant letter format propeptidecleavage domain 45  46 Elastase Amino acid, threeGlu Thr Ala Pro Gly Val Val Gly variant letter format propeptidecleavage domain 46  47 Elastase Amino acid, threeGlu Thr Asn Pro Gly Val Val Gly variant letter format propeptidecleavage domain 47  48 Elastase Amino acid, threeGlu Thr Asn Pro Ala Val Val Gly variant letter format propeptidecleavage domain 48  49 Elastase Amino acid, threeGlu Thr Asn His Ala Val Val Gly variant letter format propeptidecleavage domain 49  50 Yeast alpha- Amino acid, threeMet-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- mating factorletter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr- signal Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu- peptide,Ala-Val-Ile-Gly-Tyr-Leu-Asp-Leu-Glu-Gly-Asp-Phe- propeptide,Asp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- and spacerAsn-Asn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala- sequence 1Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Asp- Lys-Arg-Glu-Ala-Glu-Ala 51 Yeast alpha- Amino acid, threeMet-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- mating factorletter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr-signal pep- Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu- tide andAla-Val-Ile-Gly-Tyr-Leu-Asp-Leu-Glu-Gly-Asp-Phe- propeptideAsp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- sequence 2Asn-Asn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Asp-  52 ElastaseAmino acid, three Glu Thr Lys Pro Ala Val Val Gly variant letter formatpropeptide cleavage domain 52  53 Elastase Amino acid, threeGlu Thr His Pro Ala Val Val Gly variant letter format propeptidecleavage domain 53  54 Elastase Amino acid, threeGlu His Asn Pro Ala Val Val Gly variant letter format propeptidecleavage domain 54  55 Elastase Amino acid, threeHis Thr Asn Pro Ala Val Val Gly variant letter format propeptidecleavage domain 55  56 Elastase Amino acid, threePro Thr His Pro Ala Val Val Gly variant letter format propeptidecleavage domain 56  57 Elastase Amino acid, threePro Thr Asn Pro Ala Val Val Gly variant letter format propeptidecleavage domain 57  58 Elastase Amino acid, threeHis Thr His Pro Ala Val Val Gly variant letter format propeptidecleavage domain 58  59 Elastase Amino acid, threeGlu Thr Phe Pro Ala Val Val Gly variant letter format propeptidecleavage domain 59  60 Elastase Amino acid, threeHis Thr Phe Pro Ala Val Val Gly variant letter format propeptidecleavage domain 60  61 Elastase Amino acid, threeGly Thr Phe Pro Ala Val Val Gly variant letter format propeptidecleavage domain 61  62 Elastase Amino acid, threeHis Thr Gly Pro Ala Val Val Gly variant letter format propeptidecleavage domain 62  63 Elastase Amino acid, threeHis Thr Lys Pro Ala Val Val Gly variant letter format propeptidecleavage domain 63  64 Elastase Amino acid, singleTQDLPETNPAVVGGTEAGRNSWPSQISLQYRSGG proenzyme letter format, SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 48TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN  65 ElastaseAmino acid, single TQDLPETNHAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 49TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN  66 ElastaseAmino acid, single TQDLPETKPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 52TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN  67 ElastaseAmino acid, single TQDLPETHPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 53TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN  68 ElastaseAmino acid, single TQDLPEHNPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 54TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN  69 ElastaseAmino acid, single TQDLPHTNPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SRYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage X = V or LDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 55TGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYIS WINNVIASN  70 Wild-typeAmino acid, single ARVVGGTEAGRNSWPSQISLQYRSGGSRYHTCG elastase +letter format,  GTLIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQN AlaArg wherein:DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA cleavage X = V or LQSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT variantKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVK NTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIA SN  71 Wild-type Amino acid, singleRVVGGTEAGRNSWPSQISLQYRSGGSRYHTCGG elastase + letter format, TLIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQND Arg cleavage wherein:GTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQ variant X = V or LSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKTK TNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG VTSFVSSRGCNVSRKPTVFTQVSAYISWINNVIASN 72 Variant 48 Amino acid, three Thr Gln Asp Leu Pro Glu Thr Asn Pro Alahuman letter format elastase activation peptide  73 Variant 55Amino acid, three Thr Gln Asp Leu Pro His Thr Asn Pro Ala humanletter format elastase activation peptide  74 Human Amino acid, threeXaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ elastase letter format Xaa₁ =glutamate, histidine, proline, glycine, cleavageasparagine, lysine, or alanine domain Xaa₂ =threonine, alanine, proline or histidine consensus Xaa₃ =alanine, leucine, isoleucine, methionine, sequence;lysine, asparagine or valine corresponds Xaa₄ =proline, alanine, leucine, isoleucine,  to residuesglycine, valine, or threonine P5, P4, P3, Xaa₅ =alanine, leucine, valine,  P2, P1, P′1, isoleucine, or serine P′2, and P′3  Xaa₆ = alanine, leucine, valine, of an elastaseisoleucine or serine  cleavage Xaa₇ = glycine, alanine, or valine domainXaa₈ = valine, threonine, phenylalanine, tyrosine, or tryptophan  75 PCRNucleic Acid ATC TAC GTA GTC GGA GGG ACT GAG GCC mutagenesis primer  76PCR Nucleic Acid gtc gac aag ctt atc agt tgg agg cga t mutagenesisprimer  77 Mature ELA1 Protein, single VVGGTEAGRNSWPSQISLQYRSGGSRYHTCGGTL C-terminal letter formatIRQNWVMTAAHCVDYQKTFRVVAGDHNLSQNDGT variant ofEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV Talas et al.TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN GQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVRSGCQGDSGGPPPLLGEWQVFSPW SDQLCVQPGL  78 Mature ELA-1Protein, single  VVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTL variantsletter format,  IRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGT wherein:EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV B = W or RTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN J = M or VGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM X = V or LVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV Z = Q or RTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN  79 Activation Protein, single TUDLPETNAR peptide letter format,  variants  wherein (wild-type,  U =Q or H trypsin cleavable)  80 Activation Protein, three Thr Xaa₁ Asp Leu Pro Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ peptide letter formatXaa₁ = glutamine or histidine variant Xaa₂ =glutamate, histidine, proline, glycine, consensusasparagine, lysine, or alanine Xaa₃ =threonine, alanine, proline or histidine Xaa₄ =alanine, leucine, isoleucine, methionine, lysine, asparagine or valineXaa₅ = proline, alanine, leucine, isoleucine, glycine, valine, or threonine Xaa₆ =alanine, leucine, valine, isoleucine,  or serine  81 Coding regionNucleotide ACTCAGGACCTTCCGGAAACCAATGCCCGGGTA of ELA-1.2AGTCGGAGGGACTGAGGCCGGGAGGAACTCCTG GCCCTCTCAGATTTCCCTCCAGTACCGGTCTGGAGGTTCCTGGTATCACACCTGTGGAGGGACCCT TATCAGACAGAACTGGGTGATGACAGCTGCACACTGCGTGGATTACCAGAAGACTTTCCGCGTGGT GGCTGGAGACCATAACCTGAGCCAGAATGATGGCACTGAGCAGTACGTGAGTGTGCAGAAGATCGT GGTGCATCCATACTGGAACAGCGATAACGTGGCTGCAGGCTATGACATCGCCCTGCTGCGCCTGGC CCAGAGCGTTACCCTCAATAGCTATGTCCAGCTGGGTGTTCTGCCCCAGGAGGGAGCCATCCTGGCT AACAACAGTCCCTGCTACATCACAGGCTGGGGCAAGACCAAGACCAATGGGCAGCTGGCCCAGACC TTGCAGCAGGCTTACCTGCCCTCTGTGGACTATGCCATCTGCTCCAGCTCCTCCTACTGGGGCTCC ACTGTGAAGAACACTATGGTGTGTGCTGGTGGAGATGGAGTTCGCTCTGGATGTCAGGGTGACTCT GGGGGCCCCCTCCATTGCTTGGTGAATGGCAAGTATTCTCTTCATGGAGTGACCAGCTTTGTGTCCA GCCGGGGCTGTAATGTCTCTAGAAAGCCTACAGTCTTCACACGGGTCTCTGCTTACATCTCCTGGAT AAATAATGTCATCGCCTCCAACTGATAA  82Translation Protein, single  TQDLPETNARVVGGTEAGRNSWPSQISLQYRSGGproduct of letter format SWYHTCGGTLIRQNWVMTAAHCVDYQKTFRVVAG ELA-1.2ADHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY (trypsinDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI activatedTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY pPROT24WGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV sequence)NGKYSLHGVTSFVSSRGCNVSRKPTVFTRVSAYIS WINNVIASN  83 Variants ofProtein, single  TUDLPETNARVVGGTEAGRNSWPSQISLQYRSGG translationletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG product of wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY ELA-1.2A U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI (trypsin B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY activated J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV pPROT24 X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS sequence) Z = Q or R WINNVIASN  84Mature human Amino acid, single VVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTLelastase I, letter format,  IRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGTincluding  wherein: EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV first  B =W or R TLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN “valine” J = M or VGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM X = V or LVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV Z = Q or RTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN  85 Mature human Amino acid, singleVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTLI elastase I, letter format, RQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGTE minus first wherein:QYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSVT “valine” B = W or RLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTNG J = M or VQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMV X = V or LCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVT Z = Q or RSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN  86 Mature human Amino acid, singleGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTLIR elastase I, letter format, QNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGTE minus first  wherein:QYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSVT two “valines” B = W or RLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTNG J = M or VQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTMV X = V or LCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGVT Z = Q or RSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN  87 Mature human Amino acid, singleAVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGTL elastase I, letter format, IRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDGT with first wherein:EQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQSV “valine” B = W or RTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKTN substituted  J = M or VGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNTM by “alanine” X = V or LVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHGV Z = Q or RTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN  88 Engineered Amino acid, singleTUDLPETNAAVVGGTEAGRNSWPSQISLQYRSGG elastase letter format, SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein  wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY no. 1  U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI (pPROT42 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY variant) J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN  89 EngineeredAmino acid, single TUDLPETNAAAVGGTEAGRNSWPSQISLQYRSGG elastaseletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY  no. 2 U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN  90 EngineeredAmino acid, single TUDLPETAAAVVGGTEAGRNSWPSQISLQYRSGG elastaseletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein  wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY no. 3 U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN  91 EngineeredAmino acid, single TUDLPETNNAPVGGTEAGRNSWPSQISLQYRSGG elastaseletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein  wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY no. 4 U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN  92 EngineeredAmino acid, single TUDLPETNARVVGGTEAGRNSWPSQISLQYRSGG elastaseletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG proprotein  wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY no. 5  U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI (pPROT24 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY trypsin J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV activated X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS sequence) Z = Q or R WINNVIASN  93Consensus Amino acid, three Xaa₁ Pro Xaa₂ elastase letter format Xaa₁ =alanine, leucine, isoleucine,  recognitionmethionine, lysine, asparagine or valine sequence 2 Xaa₂ =alanine, leucine, valine, isoleucine,  (Positions  or serine P3-P2-P1) 94 pPROT42 P3 Amino acid, single AAVVGGTEAGRNSWPSQISLQYRSGGSBYHTCGcleavage  letter format,  GTLIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNsite variant wherein: DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA elastase B =W or R QSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT J = M or VKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVK X = V or LNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSX Z = Q or RHGVTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIA SN  95 pPROT42 P2Amino acid, single AVVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGT cleavage letter format,  LIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDG site variant wherein:TEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQS elastase B = W or RVTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKT J = M or VNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNT X = V or LMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG Z = Q or RVTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN  96 Yeast alpha- Amino acid, threeMet-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- mating factorletter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr- signal Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu- peptide,Ala-Val-Ile-Gly-Tyr-Ser-Asp-Leu-Glu-Gly-Asp-Phe- propeptide,Asp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- and spacerAsn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-Ser- sequenceIle-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Glu-Lys- Arg-Glu-Ala-Glu-Ala  97Yeast alpha- Amino acid, threeMet-Arg-Phe-Pro-Ser-Ile-Phe-Thr-Ala-Val-Leu-Phe- mating factorletter format Ala-Ala-Ser-Ser-Ala-Leu-Ala-Ala-Pro-Val-Asn-Thr-signal peptide Thr-Thr-Glu-Asp-Glu-Thr-Ala-Gln-Ile-Pro-Ala-Glu- andAla-Val-Ile-Gly-Tyr-Ser-Asp-Leu-Glu-Gly-Asp-Phe- propeptideAsp-Val-Ala-Val-Leu-Pro-Phe-Ser-Asn-Ser-Thr-Asn- sequenceAsn-Gly-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Glu-  98 ElastaseAmino acid, single TUDLPETNPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 48 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN  99 ElastaseAmino acid, single TUDLPETNHAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 49 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN 100 ElastaseAmino acid, single TUDLPETKPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 52 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN 101 ElastaseAmino acid, single TUDLPETHPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 53 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN 102 ElastaseAmino acid, single TUDLPEHNPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 54 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN 103 ElastaseAmino acid, single TUDLPHTNPAVVGGTEAGRNSWPSQISLQYRSGG proenzymeletter format,  SBYHTCGGTLIRQNWVJTAAHCVDYQKTFRVVAG with variant wherein:DHNLSQNDGTEQYVSVQKIVVHPYWNSDNVAAGY cleavage U = Q or HDIALLRLAQSVTLNSYVQLGVLPQEGAILANNSPCYI domain 55 B = W or RTGWGKTKTNGQLAQTLQQAYLPSVDYAICSSSSY J = M or VWGSTVKNTMVCAGGDGVRSGCQGDSGGPLHCLV X = V or LNGKYSXHGVTSFVSSRGCNVSRKPTVFTZVSAYIS Z = Q or R WINNVIASN 104 Wild-typeAmino acid, single ARVVGGTEAGRNSWPSQISLQYRSGGSBYHTCG elastase +letter format,  GTLIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQN AlaArg wherein:DGTEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLA cleavage B = W or RQSVTLNSYVQLGVLPQEGAILANNSPCYITGWGKT variant J = M or VKTNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVK X = V or LNTMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSX Z = Q or RHGVTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIA SN 105 Wild-type Amino acid, singleRVVGGTEAGRNSWPSQISLQYRSGGSBYHTCGGT elastase + letter format, LIRQNWVJTAAHCVDYQKTFRVVAGDHNLSQNDG Arg cleavage wherein:TEQYVSVQKIVVHPYWNSDNVAAGYDIALLRLAQS variant B = W or RVTLNSYVQLGVLPQEGAILANNSPCYITGWGKTKT J = M or VNGQLAQTLQQAYLPSVDYAICSSSSYWGSTVKNT X = V or LMVCAGGDGVRSGCQGDSGGPLHCLVNGKYSXHG Z = Q or RVTSFVSSRGCNVSRKPTVFTZVSAYISWINNVIASN 106 Mature human Amino acid, singleTEAGRNSWPSQISLQYRSGGSBYHTCGGTLIRQN elastase I, letter format, WVJTAAHCVDYQKTFRVVAGDHNLSQNDGTEQYV minus N wherein:SVQKIVVHPYWNSDNVAAGYDIALLRLAQSVTLNS terminal B = W or RYVQLGVLPQEGAILANNSPCYITGWGKTKTNGQLA “VVGG” J = M or VQTLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAG sequence X = V or LGDGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFV (SEQ ID Z = Q or RSSRGCNVSRKPTVFTZVSAYISWINNVIASN NO: 127) 107 Mature humanAmino acid, single AGRNSWPSQISLQYRSGGSBYHTCGGTLIRQNWV elastase I,letter format,  JTAAHCVDYQKTFRVVAGDHNLSQNDGTEQYVSV minus N wherein:QKIVVHPYWNSDNVAAGYDIALLRLAQSVTLNSYV terminal B = W or RQLGVLPQEGAILANNSPCYITGWGKTKTNGQLAQT “VVGGTE” J = M or VLQQAYLPSVDYAICSSSSYWGSTVKNTMVCAGGD sequence X = V or LGVRSGCQGDSGGPLHCLVNGKYSXHGVTSFVSS (SEQ ID Z = Q or RRGCNVSRKPTVFTZVSAYISWINNVIASN NO: 128) 108 Mature humanAmino acid, single NSWPSQISLQYRSGGSBYHTCGGTLIRQNWVJTA elastase I,letter format,  AHCVDYQKTFRVVAGDHNLSQNDGTEQYVSVQKI minus N wherein:VVHPYWNSDNVAAGYDIALLRLAQSVTLNSYVQLG terminal B = W or RVLPQEGAILANNSPCYITGWGKTKTNGQLAQTLQQ “VVGGTEAG J = M or VAYLPSVDYAICSSSSYWGSTVKNTMVCAGGDGVR R” sequence X = V or LSGCQGDSGGPLHCLVNGKYSXHGVTSFVSSRGC (SEQ ID Z = Q or RNVSRKPTVFTZVSAYISWINNVIASN NO: 129) 109 FIG. 1A Nucleic AcidGAATTCAGTACTCAGGACCTTCCGGAAACCAATG sequenceCCCGGGTAGTCGGAGGGACTGAGGCCGGGAGG AACTCCTGGCCCTCTCAGATTTCCCTCCAGTACCGGTCTGGAGGTTCCTGGTATCACACCTGTGGAG GGACCCTTATCAGACAGAACTGGGTGATGACAGCTGCACACTGCGTGGATTACCAGAAGACTTTCC GCGTGGTGGCTGGAGACCATAACCTGAGCCAGAATGATGGCACTGAGCAGTACGTGAGTGTGCAGA AGATCGTGGTGCATCCATACTGGAACAGCGATAACGTGGCTGCAGGCTATGACATCGCCCTGCTGC GCCTGGCCCAGAGCGTTACCCTCAATAGCTATGTCCAGCTGGGTGTTCTGCCCCAGGAGGGAGCCA TCCTGGCTAACAACAGTCCCTGCTACATCACAGGCTGGGGCAAGACCAAGACCAATGGGCAGCTGG CCCAGACCTTGCAGCAGGCTTACCTGCCCTCTGTGGACTATGCCATCTGCTCCAGCTCCTCCTACTG GGGCTCCACTGTGAAGAACACTATGGTGTGTGCTGGTGGAGATGGAGTTCGCTCTGGATGTCAGGG TGACTCTGGGGGCCCCCTCCATTGCTTGGTGAATGGCAAGTATTCTCTTCATGGAGTGACCAGCTTT GTGTCCAGCCGGGGCTGTAATGTCTCTAGAAAGCCTACAGTCTTCACACGGGTCTCTGCTTACATCT CCTGGATAAATAATGTCATCGCCTCCAACTGATAAGCTTGGATCCGTCGAC 110 FIG. 1A Amino Acid, singleMKRILAIHQAMEGAPRVTLTSRANSISTSTHHSVLH sequence letter formatSGAPVGGAGADGIVHRGQVSLLQGLGQLPIGLGLA PACDVAGTVVSQDGSLLGQNTQLDIAIEGNALGQAQQGDVIACSHVIAVPVWMHHDLLHTHVLLSAIILAQ VMVSSHHAESLLVIHAVCSCHHPVLSDKGPSTGVIPGTSRPVLEGNLRGPGVPPGLSPSDYPGIGFRKVL S 111 FIG. 1B Nucleic AcidACTATTGCCAGCATTGCTGCTAAAGAAGAAGGG sequenceGTATCTCTCGAGAAAAGAGAGGCTGAAGCTACT CAGGACCTTCCGGAAACCAATGCCCGGGTAGTCGGGGGG 112 FIG. 1B Amino Acid, threeTHR ILE ALA SER ILE ALA ALA LYS GLU GLU GLY sequence letter formatVAL SER LEU GLU LYS ARG GLU ALA GLU ALATHR GLN ASP LEU PRO GLU THR ASN ALA ARG VAL VAL GLY GLY 113 FIG. 13Nucleic Acid CCGCGGACCCAGGACTTTCCAGAAACCAACGCC sequenceCGGGTAGTTGGAGGGACCGAGGCTCAGAGGAA TTCTTGGCCATCTCAGATTTCCCTCCAGTACCGGTCTGGAAGTTCGTGGGCTCACACCTGTGGAGGG ACCCTCATCAGGCAGAACTGGGTGATGACAGCCGCTCACTGCGTGGACAGAGAGTTGACCTTCCGT GTGGTGGTTGGAGAGCACAACCTGAACCAGAACGATGGCACCGAGCAGTACGTGGGGGTGCAGAA GATCGTGGTGCATCCCTACTGGAACACCGACGACGTGGCTGCAGGCTATGACATCGCCCTGCTGCG CCTGGCCCAGAGTGTAACCCTCAACAGCTACGTCCAGCTGGGTGTTCTGCCAAGGGCTGGGACCAT CCTGGCTAACAACAGTCCCTGCTACATCACAGGGTGGGGCCTGACCAGGACCAATGGGCAGCTGG CCCAGACCCTGCAGCAGGCTTACCTGCCCACCGTGGACTACGCCATCTGCTCCAGCTCCTCGTACT GGGGCTCCACCGTGAAGAACAGCATGGTGTGCGCCGGAGGGGACGGAGTTCGCTCTGGATGTCAG GGTGATTCTGGGGGCCCCCTTCATTGCTTGGTGAATGGTCAGTATGCTGTCCACGGTGTAACCAGCT TCGTGTCCCGCCTGGGCTGTAATGTCACCAGGAAGCCCACAGTCTTCACCAGGGTCTCTGCTTACAT CTCTTGGATAAATAACGTCATTGCCAGCAACTGATAATCTAGA 114 FIG. 14 Amino Acid, singleTQDFPETNARVVGGTEAQRNSWPSQISLQYRSGS sequence letter formatSWAHTCGGTLIRQNVVVMTAAHCVDRELTFRVVVG EHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIALLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYI TGWGLTRTNGQLAQTLQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGPLHCLV NGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 115 Cleavage Amino Acid, threePhe Pro Glu Thr Asn Ala Arg Val Val Gly domain letter format sequence oftrypsin- activated pPROT101- 24-V 116 Cleavage Amino Acid, threePhe Pro Glu Thr Asn Ala Ala Val Val Gly domain letter format sequence ofauto-activated pPROT101- 42-V 117 Cleavage Amino Acid, threeLeu Pro His Thr Asn Pro Ala Val Val Gly domain letter format sequence ofauto-activated pPROT101- 49-V 118 Cleavage Amino Acid, threePhe Pro Glu Thr Asn His Ala Val Val Gly domain letter format sequence ofauto-activated pPROT101- 55L-V 119 Consensus Amino acid, threeXaa₁ Xaa₂ Xaa₃ elastase letter format Xaa₁ =any natural amino acid except pro or gly recognition Xaa₂ =pro, ala, leu, ile, gly, val, his or thr sequence 5 Xaa₃ =ala, leu, val, ile, or ser (Positions Xaa₁ = P3, Xaa₂ = P2, Xaa₃ = P1)120 Consensus Amino acid, three Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ sequence 1letter format Xaa₁ = any natural amino acid for the Xaa₂ =any natural amino acid except gly, lys, propeptide phe, tyr, trp, or argportion of  Xaa₃ = any natural amino acid except pro or gly the cleavageXaa₄ = pro, ala, leu, ile, gly, val, his or thr domain Xaa₅ =ala, leu, val, ile, or ser (Positions Xaa₁ = P5, Xaa₂ = P4, Xaa₃ = P3,Xaa₄ = P2, Xaa₅ = P1) 121 Consensus Amino acid, threeXaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ sequence 2 letter formatXaa₁₀ for the Xaa₁ = thr activation Xaa₂ = gln or his peptide Xaa₃ = asp(Positions Xaa₄ = leu Xaa₁ = P10, Xaa₅ = pro Xaa₂ = P9, Xaa₆ =any natural amino acid Xaa₃ = P8, Xaa₇ =any natural amino acid except gly, lys,  Xaa₄ = P7,phe, tyr, trp, or arg Xaa₅ = P6, Xaa₈ =any natural amino acid except pro or gly Xaa₆ = P5, Xaa₉ =pro, ala, leu, ile, gly, val, his or thr Xaa₇ = P4, Xaa₁₀ =ala, leu, val, ile, or ser Xaa₈ = P3, Xaa₉ = P2, Xaa₁₀ = P1) 122Proelastase Amino acid, threeXaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ consensus letter formatXaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ sequence 1 Xaa₁ = thr for residues Xaa₂ =gln or his P10 through Xaa₃ = asp P3′ Xaa₄ = leu Xaa₅ = pro Xaa₆ =any natural amino acid Xaa₇ = any natural amino acid except gly, lys, phe, tyr, trp, or arg Xaa₈ = any natural amino acid except pro or glyXaa₉ = pro, ala, leu, ile, gly, val, his or thr Xaa₁₀ =ala, leu, val, ile, or ser Xaa₁₁ = ala, leu, val, ile, or ser Xaa₁₂ =gly, ala, or val Xaa₁₃ = gly, val, thr, phe, tyr, or trp 123 ProelastaseAmino acid, three Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ cleavageletter format Xaa₁ = glu, his, pro, gly, asn, lys, or ala domain Xaa₂ =thr, ala, pro or his consensus Xaa₃ =ala, leu, ile, met, lys, asn or val sequence 2 Xaa₄ =pro, ala, leu, ile, gly, val, or thr (Positions Xaa₅ =ala, leu, val, ile, or ser Xaa₁ = P5, Xaa₆ = ala, leu, val, ile, or serXaa₂ = P4, Xaa₇ = gly, ala, or val Xaa₃ = P3, Xaa₈ =gly, val, thr, phe, tyr, or trp Xaa₄ = P2, Xaa₅ = P1, Xaa₆ = P1′, Xaa₇ =P2′, Xaa₈ = P3′) 124 Consensus Amino acid, three Xaa₁ Xaa₂ Xaa₃ elastaseletter format Xaa₁ = ala, leu, ile, met, lys, asn, his, or valrecognition Xaa₂ = pro, ala, leu, ile, gly, val, or thr sequence 6Xaa₃ = ala, leu, val, ile, or ser (Positions P3- P2-P1) 125 ProelastaseAmino acid, three Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ cleavageletter format Xaa₁ = glu, his, pro, gly, asn, lys, or ala domain Xaa₂ =thr, ala, pro or his consensus Xaa₃ =ala, leu, ile, met, lys, asn, thr, or val sequence 3 Xaa₄ =pro, ala, leu, ile, gly, val, asn, or thr (Positions Xaa₅ =ala, leu, val, ile, asn, or ser Xaa₁ = P5, Xaa₆ =ala, leu, val, ile, or ser Xaa₂ = P4, Xaa₇ = gly, ala, or val Xaa₃ = P3,Xaa₈ = gly, val, thr, phe, tyr, or trp Xaa₄ = P2, Xaa₅ = P1, Xaa₆ = P1′,Xaa₇ = P2′, Xaa₈ = P3′)

8. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES

The present invention is exemplified by the specific embodiments below.

In a 1^(st) embodiment, the disclosure provides a protein comprising (i)an elastase activation sequence comprising an elastase recognitionsequence operably linked to (ii) the amino acid sequence of a matureelastase.

In a 2^(nd) embodiment, the disclosure provides a protein of the 1^(st)embodiment, wherein the elastase recognition sequence comprises SEQ IDNO:11.

In a 3^(rd) embodiment, the disclosure provides a protein of the 1^(st)embodiment, wherein the elastase recognition sequence comprises SEQ IDNO:12.

In a 4^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment, wherein the elastase recognition sequence comprises SEQ IDNO:13.

In a 5^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment, wherein the elastase recognition sequence comprises SEQ IDNO:93.

In a 6^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment, 2^(nd) embodiment or 4^(th) embodiment, wherein the elastaserecognition sequence comprises any one of SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21.

In a 7^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment, wherein the activation sequence comprises SEQ ID NO:80.

In an 8^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment or the 7^(th) embodiment, wherein the activation sequencecomprises SEQ ID NO:23, SEQ ID NO:72, or SEQ ID NO:73.

In a 9^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment, wherein the protein comprises a cleavage domain comprisingSEQ ID NO:74.

In a 10^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment or 9^(th) embodiment, wherein the cleavage domain comprisesany one of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, SEQID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55.

In an 11^(th) embodiment, the disclosure provides a protein of any oneof the 1^(st)-5^(th) or 7^(th) embodiments which comprises SEQ ID NO:64.

In a 12^(th) embodiment, the disclosure provides a protein of any one ofthe 1^(st)-5^(th) or 7^(th) embodiments which comprises SEQ ID NO:69.

In a 13^(th) embodiment, the disclosure provides a type I proelastaseprotein comprising a cleavage domain sequence of SEQ ID NO:74.

In a 14^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 13^(th) embodiment, wherein the cleavage domain comprisesany one of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, SEQID NO:52, SEQ ID NO:53, SEQ ID NO:54 or SEQ ID NO:55.

In a 15^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 13^(th) embodiment or 14^(th) embodiment, wherein themature elastase sequence comprises a sequence having at least 85%sequence identity to the amino acid sequence from position 6 (e.g.,C-terminal to the P5′ residue according to the elastase amino aciddesignations herein) to the end of SEQ ID NO:84 or SEQ ID NO:1.

In a 16^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 13^(th) embodiment or 14^(th) embodiment, which comprisesa sequence having at least 95% sequence identity to the amino acidsequence from position 6 (e.g., C-terminal to the P5′ residue accordingto the elastase amino acid designations herein) to the end of SEQ IDNO:84 or SEQ ID NO:1.

In a 17^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 13^(th) embodiment or 14^(th) embodiment, which comprisesa sequence having at least 98% sequence identity to the amino acidsequence from position 6 to the end of SEQ ID NO:84 or SEQ ID NO:1.

In a 18^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 13^(th) embodiment or 14^(th) embodiment, wherein themature elastase comprises a sequence having up to 10 conservative aminoacid changes relative to the amino acid sequence from position 6 to theend of SEQ ID NO:84 or SEQ ID NO:1.

In a 19^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 13^(th) embodiment or 14^(th) embodiment, which comprisesa sequence having up to 7 conservative amino acid changes relative tothe amino acid sequence from position 6 to the end of SEQ ID NO:84 orSEQ ID NO:1.

In a 20^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 13^(th) embodiment or 14^(th) embodiment, which comprisesa sequence having up to 5 conservative amino acid changes relative tothe amino acid sequence from position 6 to the end of SEQ ID NO:84 orSEQ ID NO:1.

In a 21^(st) embodiment, the disclosure provides a type I proelastaseprotein of any one of the 13^(th) to the 20^(th) embodiments, whereinthe amino acid residue denoted by “Xaa₁” in SEQ ID NO:74 is glutamate orhistidine.

In a 22^(nd) embodiment, the disclosure provides a type I proelastaseprotein of any one of the 13^(th) to the 21^(st) embodiments, whereinthe amino acid residue denoted by “Xaa₄” in SEQ ID NO:74 is proline.

In a 23^(rd) embodiment, the disclosure provides a type I proelastaseprotein of any one of the 13^(th) to the 22^(nd) embodiments, whereinthe amino acid residue denoted by “Xaa₅” in SEQ ID NO:74 is alanine.

In a 24^(th) embodiment, the disclosure provides a type I proelastaseprotein of any one of the 13^(th) to 23^(rd) embodiments, wherein theamino acid residue denoted by “Xaa₁” in SEQ ID NO:74 is histidine, by“Xaa₄” in SEQ ID NO:74 is proline, and by “Xaa₅” in SEQ ID NO:74 isalanine.

In a 25^(th) embodiment, the disclosure provides a type I proelastaseprotein of any one of the 13^(th) to 24^(th) embodiments which comprisesthe amino acid sequence of SEQ ID NO:103.

In a 26^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 25^(th) embodiment which comprises the amino acidsequence of SEQ ID NO:64.

In a 27^(th) embodiment, the disclosure provides a type I proelastaseprotein of the 25^(th) embodiment which comprises the amino acidsequence of SEQ ID NO:69.

In a 28^(th) embodiment, the disclosure provides a protein of any one ofthe 1^(st) to 27^(th) embodiments which is isolated.

In a 29^(th) embodiment, the disclosure provides a protein of any one ofthe 1^(st) to 27^(th) embodiments which comprises a signal sequence.

In a 30^(th) embodiment, the disclosure provides a protein comprising(i) a signal sequence; (ii) an elastase activation sequence comprisingan elastase recognition sequence; and (iii) the amino acid sequence of amature elastase.

In a 31^(st) embodiment, the disclosure provides a protein of the30^(th) embodiment, wherein the signal sequence is operable in Pichiapastoris.

In a 32^(nd) embodiment, the disclosure provides a protein of the30^(th) embodiment, wherein the signal sequence is a yeast α-factorsignal peptide.

In a 33^(rd) embodiment, the disclosure provides a protein of the32^(rd) embodiment, wherein the yeast α-factor signal peptide comprisesthe amino acid sequence of SEQ ID NO:34.

In a 34^(th) embodiment, the disclosure provides a protein of the30^(th) embodiment, wherein the signal sequence is a mammalian secretionsignal sequence.

In a 35^(th) embodiment, the disclosure provides a protein of the34^(th) embodiment, wherein the mammalian secretion signal sequence is aporcine type I elastase signal sequence.

In a 36^(th) embodiment, the disclosure provides a protein of the34^(th) embodiment, wherein the mammalian secretion signal sequence is ahuman type I elastase signal sequence.

In a 37^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment or the 30^(th) embodiment wherein the elastase recognitionsequence is a type I human elastase recognition sequence.

In a 38^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment or the 30^(th) embodiment wherein the mature elastase is ahuman type I elastase.

In a 39^(th) embodiment, the disclosure provides a protein of the 1^(st)embodiment or the 39^(th) embodiment wherein the mature elastase is aporcine type I elastase.

In a 40^(th) embodiment, the disclosure provides a nucleic acid encodinga protein of any one of the 1^(st) to 30^(th) embodiments.

In a 41^(st) embodiment, the disclosure provides a nucleic acid moleculecomprising a nucleotide sequence that encodes a protein, said proteincomprising (i) an activation sequence comprising an elastase recognitionsequence operably linked to (ii) the amino acid sequence of an elastase.

In a 42^(nd) embodiment, the disclosure provides a nucleic acid moleculeof the 41^(st) embodiment, wherein the protein further comprises asignal sequence operably linked to said activation sequence.

In a 43^(rd) embodiment, the disclosure provides a nucleic acid moleculeof the 41^(st) embodiment or the 42^(nd) embodiment, wherein the signalsequence is operable in Pichia pastoris.

In a 44^(th) embodiment, the disclosure provides a nucleic acid of anyone of the 41^(st) to 43^(rd) embodiments, wherein the elastaserecognition sequence is a type I elastase recognition sequence.

In a 45^(th) embodiment, the disclosure provides a nucleic acid of anyone of the 41^(st) to 44^(th) embodiments, wherein the elastaserecognition sequence is a type I human elastase recognition sequence.

In a 46^(th) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 45^(th) embodiments wherein the elastaserecognition sequence comprises SEQ ID NO:11.

In a 47^(th) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 45^(th) embodiments wherein the elastaserecognition sequence comprises SEQ ID NO:12.

In a 48^(th) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 45^(th) embodiments wherein the elastaserecognition sequence comprises SEQ ID NO:13.

In a 49^(th) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 45^(th) embodiments wherein the elastaserecognition sequence comprises SEQ ID NO:93.

In a 50^(th) embodiment, the disclosure provides a nucleic acid of anyone of the 41^(st) to 46^(th) and 48^(th) embodiments, wherein theelastase recognition sequence comprises any one of SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21.

In a 51^(st) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 50^(th) embodiments, wherein the activationsequence comprises SEQ ID NO:80.

In a 52^(nd) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 45^(th) and 51^(st) embodiments, whereinthe activation sequence comprises SEQ ID NO:23, SEQ ID NO:72, or SEQ IDNO:73.

In a 53^(rd) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 46^(th) embodiments, wherein the proteincomprises a cleavage domain comprising SEQ ID NO:74.

In a 54^(th) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 46^(th) and 53^(rd) embodiments, whereinthe cleavage domain comprises any one of SEQ ID NO:42, SEQ ID NO:43, SEQID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and SEQID NO:55.

In a 55^(th) embodiment, the disclosure provides a nucleic acid of anyone of the 41^(st) to 54^(th) embodiments, wherein the elastase is amature human type I elastase.

In a 56^(th) embodiment, the disclosure provides a nucleic acid of anyone of the 41^(st) to 54^(th) embodiments, wherein the elastase is amature porcine type I elastase.

In a 57^(th) embodiment, the disclosure provides a nucleic acid of anyone of the 41^(st) to 54^(th) embodiments, wherein the elastasecomprises the amino acid sequence of any one of SEQ ID NO:5, SEQ IDNO:84, SEQ ID NO:87, and SEQ ID NO:39.

In a 58^(th) embodiment, the disclosure provides a nucleic acid of the57^(th) embodiment wherein the elastase comprises the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:4.

In a 59^(th) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 42^(nd) to 58^(th) embodiments, wherein the signalsequence is a yeast α-factor signal peptide.

In a 60^(th) embodiment, the disclosure provides a nucleic acid moleculeof the 59^(th) embodiment, wherein the protein comprises the amino acidsequence of any one of SEQ ID NO:34, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:96 and SEQ ID NO:97.

In a 61^(st) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 42^(nd) and 44^(th) to 58^(th) embodiments, whereinthe signal sequence is a mammalian secretion signal sequence.

In a 62^(nd) embodiment, the disclosure provides a nucleic acid moleculeof the 61^(st) embodiment wherein the mammalian secretion signalsequence is a porcine type I elastase signal sequence.

In a 63^(rd) embodiment, the disclosure provides a nucleic acid moleculeof the 61^(st) embodiment wherein the mammalian secretion signalsequence is a human type I elastase signal sequence.

In a 64^(th) embodiment, the disclosure provides a nucleic acid moleculeof any one of the 41^(st) to 63^(rd) embodiments, wherein the proteincomprises the amino acid sequence of any one of SEQ ID NOS.: 88 to 91and 98 to 103.

In a 65^(th) embodiment, the disclosure provides a nucleic acid moleculeof the 64^(th) embodiment, wherein the protein comprises the amino acidsequence of any one of SEQ ID NOS.: 6 to 9 and 64 to 69.

In a 66^(th) embodiment, the disclosure provides a nucleic acid moleculeof the 64^(th) embodiment, wherein the protein comprises any of thecombinations of elastase polymorphisms set forth in Table 2.

In a 67^(th) embodiment, the disclosure provides a nucleic acid moleculeof the 64^(th) embodiment, wherein the protein comprises any of thecombinations of elastase polymorphisms set forth in Table 3.

In a 68^(th) embodiment, the disclosure provides a protein encoded bythe nucleic acid molecule of any one of the 41^(st) to 67^(th)embodiments.

In a 69^(th) embodiment, the disclosure provides a protein of the68^(th) embodiment which is isolated.

In a 70^(th) embodiment, the disclosure provides a vector comprising thenucleic acid molecule of any one of the 41^(st) to 67^(th) embodiments.

In a 71^(st) embodiment, the disclosure provides a vector of the 70^(th)embodiment further comprising a nucleotide sequence that controls geneexpression is operably linked to the nucleotide sequence which encodessaid protein.

In a 72^(nd) embodiment, the disclosure provides a vector of the 70^(th)embodiment or the 71^(st) embodiment in which the nucleotide sequencewhich encodes said protein is multimerized.

In a 73^(rd) embodiment, the disclosure provides a host cell comprisingthe vector of any one of the 70^(th) to 72^(nd) embodiments.

In a 74^(th) embodiment, the disclosure provides a host cell of the73^(rd) embodiment in which at least one copy of said vector isintegrated into the host cell genome.

In a 75^(th) embodiment, the disclosure provides a host cell of the74^(th) embodiment in which one copy of said vector is integrated intothe host cell genome.

In a 76^(th) embodiment, the disclosure provides a host cell of the74^(th) embodiment in which two to five copies of said vector areintegrated into the host cell genome.

In a 77^(th) embodiment, the disclosure provides a host cell of the74^(th) or 76^(th) embodiment in which two copies of said vector areintegrated into the host cell genome.

In a 78^(th) embodiment, the disclosure provides a host cell of the74^(th) or 76^(th) embodiment in which three copies of said vector areintegrated into the host cell genome.

In a 79^(th) embodiment, the disclosure provides a host cell comprisingat least one copy of the nucleic acid molecule of any one of the 41^(st)to 67^(th) embodiments integrated into its genome.

In an 80^(th) embodiment, the disclosure provides a host cell of the79^(th) embodiment in which one copy of said nucleic acid molecule isintegrated into its genome.

In an 81^(st) embodiment, the disclosure provides a host cell of the79^(th) embodiment in which two to five copies of said nucleic acidmolecule is integrated into its genome.

In an 82^(nd) embodiment, the disclosure provides a host cell of the79^(th) embodiment in which two copies of said nucleic acid molecule isintegrated into its genome.

In an 83^(rd) embodiment, the disclosure provides a host cell of the79^(th) embodiment in which three copies of said nucleic acid moleculeis integrated into its genome.

In an 84^(th) embodiment, the disclosure provides a cell geneticallyengineered to express the nucleic acid molecule of any one of the41^(st) to 67^(th) embodiments.

In an 85^(th) embodiment, the disclosure provides a cell of the 84^(th)embodiment, wherein the nucleotide sequence is operably linked to amethanol-inducible promoter.

In an 86^(th) embodiment, the disclosure provides a Pichia pastoris cellgenetically engineered to express the nucleotide sequence of any one ofthe 41^(st) to 67^(th) embodiments.

In an 87^(th) embodiment, the disclosure provides a Pichia pastoris cellof the 87^(th) embodiment, in which the nucleotide sequence is operablylinked to a methanol inducible promoter.

In an 88^(th) embodiment, the disclosure provides a cell culturesupernatant comprising the protein of any one of the 1^(st) to 27^(th)embodiments or the 68^(th) embodiment.

In an 89^(th) embodiment, the disclosure provides a method of producingan elastase protein, comprising culturing the host cell of the 84^(th)embodiment under conditions in which the protein is produced.

In a 90^(th) embodiment, the disclosure provides a method of producingan elastase protein, comprising culturing the host cell of the 85^(th)embodiment under conditions in which the protein is produced.

In a 91^(st) embodiment, the disclosure provides a method of producingan elastase protein, comprising culturing the host cell of the 86^(th)embodiment under conditions in which the protein is produced.

In a 92^(nd) embodiment, the disclosure provides a method of producingan elastase protein, comprising culturing the host cell of the 87^(th)embodiment under conditions in which the protein is produced.

In a 93^(rd) embodiment, the disclosure provides a method of the 91^(st)embodiment or the 92^(nd) embodiment, wherein said conditions include aperiod of growth or induction at a pH of 2 to 6.

In a 94^(th) embodiment, the disclosure provides a method of the 91^(st)embodiment or the 92^(nd) embodiment, wherein said conditions comprise aperiod of growth or induction at a temperature of 22° C. to 28° C.

In a 95^(th) embodiment, the disclosure provides a method of any one ofthe 89^(th) to 92^(nd) embodiments, wherein the host cell is cultured incomplex medium.

In a 96^(th) embodiment, the disclosure provides a method of the 95^(th)embodiment, wherein the complex medium is buffered methanol-complexmedium or buffered glycerol-complex medium.

In a 97^(th) embodiment, the disclosure provides a method of any one ofthe 89^(th) to 96^(th) embodiments, wherein the host cell is cultured inthe presence of a citrate, succinate or acetate compound.

In a 98^(th) embodiment, the disclosure provides a method of the 97^(th)embodiment, wherein the citrate, succinate or acetate compound is sodiumcitrate, sodium succinate or sodium acetate, respectively.

In a 99^(th) embodiment, the disclosure provides a method of the 97^(th)embodiment or the 98^(th) embodiment, wherein one citrate, succinate oracetate compound is present in said culture at a concentration of 5-50mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110mM.

In a 100^(th) embodiment, the disclosure provides a method of the97^(th) embodiment or the 98^(th) embodiment, wherein more than onecitrate, succinate or acetate compound is present in said culture, andwherein the total concentration of citrate, succinate or acetatecompounds in said solution is 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM,100-150 mM, 75-125 mM, or 90-110 mM.

In a 101^(st) embodiment, the disclosure provides a method of any one ofthe 89^(th) to 100^(th) embodiments further comprising recovering theprotein.

In a 102^(nd) embodiment, the disclosure provides a method of the95^(th) embodiment, wherein the protein is recovered by recovering thesupernatant.

In a 103^(rd) embodiment, the disclosure provides a method of the95^(th) embodiment, wherein the protein is recovered from thesupernatant.

In a 104^(th) embodiment, the disclosure provides a method of embodimentany one of the 95^(th) to 104^(th) embodiments, wherein the proteinrecovered lacks the signal sequence.

In a 105^(th) embodiment, the disclosure provides a method of embodimentany one of the 95^(th) to 104^(th) embodiments, wherein the proteinrecovered lacks both the signal sequence and the activation sequence.

In a 106^(th) embodiment, the disclosure provides a method of any one ofthe 89^(th) to 92^(nd) and 93^(rd) to 105^(th) embodiments, furthercomprising raising the pH of a solution containing the protein to a pHof 6 to 12.

In a 107^(th) embodiment, the disclosure provides a method of any one ofthe 89^(th) to 92^(nd) and 93^(rd) to 106^(th) embodiments, whichfurther comprises contacting the protein with a catalytic amount of anelastase.

In a 108^(th) embodiment, the disclosure provides a method of any one of89^(th) to 92^(nd) and 93^(rd) to 106^(th) embodiments, which furthercomprises subjecting the protein to autoactivating conditions,contacting the protein with a catalytic amount of an elastase, or both.

In a 109^(th) embodiment, the disclosure provides a method of the108^(th) embodiment, wherein the protein is subjected to autoactivationconditions.

In a 110^(th) embodiment, the disclosure provides a method of the109^(th) embodiment, wherein the protein is in the supernatant whensubjected to autoactivation conditions.

In a 111^(th) embodiment, the disclosure provides a method of producingan elastase protein, comprising:

-   -   (a) culturing a host cell capable of expressing a recombinant        proelastase protein in the presence of a first citrate,        succinate or acetate compound;    -   (b) recovering the recombinant proelastase protein from said        host cell culture; and    -   (c) optionally, exposing the recombinant proelastase protein to        activation conditions to produce a mature elastase protein.

thereby producing an elastase protein.

In a 112^(th) embodiment, the disclosure provides a method of the111^(th) embodiment, wherein the method comprises the step of exposingthe recombinant proelastase protein to activation conditions to producea mature elastase protein.

In a 113^(th) embodiment, the disclosure provides a method of the112^(th) embodiment, wherein the recombinant proelastase protein ispurified prior to said exposure to activating conditions.

In a 114^(th) embodiment, the disclosure provides a method of the113^(th) embodiment, wherein said recombinant proelastase protein ispurified in the presence of a second citrate, succinate or acetatecompound, such that a solution comprising said purified proelastaseprotein and second citrate, succinate or acetate compound is produced.

In a 115^(th) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 114^(th) embodiments, wherein said first citrate,succinate or acetate compound is sodium citrate, sodium succinate orsodium acetate, respectively.

In a 116^(th) embodiment, the disclosure provides a method of the114^(th) embodiment or 115^(th) embodiment, wherein said second citrate,succinate or acetate compound is sodium citrate, sodium succinate orsodium acetate, respectively.

In a 117^(th) embodiment, the disclosure provides a method of any one ofthe 114^(th) to 116^(th) embodiments, wherein said first and secondcitrate, succinate or acetate compound are the same.

In a 118^(th) embodiment, the disclosure provides a method of any one ofthe 114^(th) to 116^(th) embodiments, wherein said first and secondcitrate, succinate or acetate compound are different.

In a 119^(th) embodiment, the disclosure provides a method of any one ofthe 114^(th) to 118^(th) embodiments, wherein one citrate, succinate oracetate compound is present in said culture at a concentration of 5-50mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110mM.

In a 120^(th) embodiment, the disclosure provides a method of any one ofthe 114^(th) to 118^(th) embodiments, wherein more than one citrate,succinate or acetate compound is present in said culture, and whereinthe total concentration of citrate, succinate or acetate compounds insaid culture is 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM,75-125 mM, or 90-110 mM.

In a 121^(st) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 120^(th) embodiments, wherein one citrate, succinate oracetate compound is present in said solution at a concentration of 5-50mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM, 75-125 mM, or 90-110mM.

In a 122^(nd) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 120^(th) embodiments, wherein more than one citrate,succinate or acetate compound is present in said solution, and whereinthe total concentration of citrate, succinate or acetate compounds insaid solution is 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM,75-125 mM, or 90-110 mM.

In a 123^(rd) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 122^(nd) embodiments, wherein the host cell is culturedin complex medium.

In a 124^(th) embodiment, the disclosure provides a method of the123^(rd) embodiment, wherein the complex medium is bufferedmethanol-complex medium or buffered glycerol-complex medium.

In a 125^(th) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 124^(th) embodiments wherein the proelastase protein isrecovered from the supernatant of said host cell.

In a 126^(th) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 125^(th) embodiments, wherein said activation conditionscomprise exposure to trypsin.

In a 127^(th) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 125^(th) embodiments, wherein said activation conditionsare autoactivation conditions.

In a 128^(th) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 127^(th) embodiments which further comprises the step ofisolating said mature elastase protein.

In a 129^(th) embodiment, the disclosure provides a method of any one ofthe 111^(th) to 128^(th) embodiments, wherein the mature elastaseprotein is a mature type I elastase protein.

In a 130^(th) embodiment, the disclosure provides a method of the129^(th) embodiment, wherein the mature type I elastase protein is ahuman type I mature elastase protein.

In a 131^(st) embodiment, the disclosure provides a method of the129^(th) embodiment, wherein the mature type I elastase protein is aporcine type I mature elastase protein.

In a 132^(nd) embodiment, the disclosure provides a method of producinga mature elastase protein, comprising:

-   -   (a) lyophilizing a proelastase protein;    -   (b) storing the lyophilized proelastase protein;    -   (c) reconstituting the lyophilized proelastase protein; and    -   (d) activating the reconstituted proelastase protein,

thereby producing a mature elastase protein.

In a 133^(rd) embodiment, the disclosure provides a method of the132^(nd) embodiment, wherein the proelastase protein is recombinant.

In a 134^(th) embodiment, the disclosure provides a method of the133^(rd) embodiment, wherein the proelastase protein is made by orobtainable by a process comprising (i) culturing a host cell that iscapable of expressing the proelastase protein under conditions in whichthe proelastase protein is expressed; and (ii) recovering theproelastase protein.

In a 135^(th) embodiment, the disclosure provides a method of the134^(th) embodiment, wherein the host cell is cultured in complexmedium.

In a 136^(th) embodiment, the disclosure provides a method of the135^(th) embodiment, wherein the complex medium is bufferedmethanol-complex medium or buffered glycerol-complex medium.

In a 137^(th) embodiment, the disclosure provides a method of any one ofthe 132^(nd) to 136^(th) embodiments, wherein the host cell is culturedin the presence of a citrate, succinate or acetate compound.

In a 138^(th) embodiment, the disclosure provides a method of the137^(th) embodiment, wherein the citrate, succinate or acetate compoundis sodium citrate, sodium succinate or sodium acetate, respectively.

In a 139^(th) embodiment, the disclosure provides a method of the137^(th) embodiment or 138^(th) embodiment, wherein one citrate,succinate or acetate compound is present in said culture at aconcentration of 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM,75-125 mM, or 90-110 mM.

In a 140^(th) embodiment, the disclosure provides a method of the137^(th) embodiment or 138^(th) embodiment, wherein more than onecitrate, succinate or acetate compound is present in said culture, andwherein the total concentration of citrate, succinate or acetatecompounds in said solution is 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM,100-150 mM, 75-125 mM, or 90-110 mM.

In a 141^(st) embodiment, the disclosure provides a method of any one ofthe 132^(st) to 140^(th) embodiments wherein the proelastase protein isrecovered from the supernatant of said host cell.

In a 142^(st) embodiment, the disclosure provides a method of any one ofthe 132^(st) to 141^(st) embodiments, wherein the proelastase protein ispurified prior to lyophilization.

In a 143^(rd) embodiment, the disclosure provides a method of any one ofthe 132^(nd) to 142^(nd) embodiments, wherein the lyophilizedproelastase protein is stored for a period of at least one day, at leastone week, at least one month or at least three months.

In a 144^(th) embodiment, the disclosure provides a method of any one ofthe 132^(nd) to 143^(rd) embodiments, wherein the proelastase protein isstored at a temperature of −80° C. to +4° C.

In a 145^(th) embodiment, the disclosure provides a method of any one ofthe 132^(st) to 144^(th) embodiments, wherein said activating stepcomprises trypsin activation.

In a 146^(th) embodiment, the disclosure provides a method of any one ofthe 132^(st) to 144^(th) embodiments, wherein said activating stepcomprises autoactivation.

In a 147^(th) embodiment, the disclosure provides a method of any one ofthe 132^(nd) to 146^(th) embodiments which further comprises the step ofisolating said mature elastase protein.

In a 148^(th) embodiment, the disclosure provides a method of any one ofthe 132^(nd) to 147^(th) embodiments, wherein the mature elastaseprotein is a mature type I elastase protein.

In a 149^(th) embodiment, the disclosure provides a method of the148^(th) embodiment, wherein the mature type I elastase protein is ahuman type I mature elastase protein.

In a 150^(th) embodiment, the disclosure provides a method of the148^(th) embodiment, wherein the mature type I elastase protein is aporcine type I mature elastase protein.

In a 151^(st) embodiment, the disclosure provides a method of producinga mature type I elastase protein comprising subjecting a recombinantautoactivated type I proelastase protein to autoactivation conditions,contacting a recombinant autoactivated type I proelastase protein with acatalytic amount of elastase, or both, thereby producing a mature type Ielastase protein.

In a 152^(nd) embodiment, the disclosure provides a method of the151^(st) embodiment, wherein said recombinant autoactivated type Iproelastase protein is obtained by or obtainable by a processcomprising:

-   -   (a) culturing the host cell of the 84^(th) or 86^(th) embodiment        under conditions in which the protein is expressed; and    -   (b) recovering the expressed protein,

thereby producing a recombinant autoactivated type I proelastaseprotein.

In a 153^(rd) embodiment, the disclosure provides a method of the152^(nd) embodiment, wherein the host cell is cultured in complexmedium.

In a 154^(th) embodiment, the disclosure provides a method of the153^(rd) embodiment, wherein the complex medium is bufferedmethanol-complex medium or buffered glycerol-complex medium.

In a 155^(th) embodiment, the disclosure provides a method of any one ofthe 152^(nd) to 154^(th) embodiments, wherein the host cell is culturedin the presence of a citrate, succinate or acetate compound.

In a 156^(th) embodiment, the disclosure provides a method of the155^(th) embodiment, wherein the citrate, succinate or acetate compoundis sodium citrate, sodium succinate or sodium acetate, respectively.

In a 157^(th) embodiment, the disclosure provides a method of the155^(th) embodiment or 156^(th) embodiment, wherein one citrate,succinate or acetate compound is present in said culture at aconcentration of 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM,75-125 mM, or 90-110 mM.

In a 158^(th) embodiment, the disclosure provides a method of the155^(th) embodiment or 156^(th) embodiment, wherein more than onecitrate, succinate or acetate compound is present in said culture, andwherein the total concentration of citrate, succinate or acetatecompounds in said solution is 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM,100-150 mM, 75-125 mM, or 90-110 mM.

In a 159^(th) embodiment, the disclosure provides a method of any one ofthe 152^(nd) to 158^(th) embodiments, wherein recovering the expressedprotein comprises recovering the supernatant.

In a 160^(th) embodiment, the disclosure provides a method of the159^(th) embodiment, wherein said autoactivation step is performed inthe supernatant.

In a 161^(st) embodiment, the disclosure provides a method of any one ofthe 152^(st) to 154^(th) embodiments, wherein the expressed protein isrecovered from the supernatant.

In a 162^(st) embodiment, the disclosure provides a method of the161^(st) embodiment, wherein said autoactivation step is performed inthe supernatant.

In a 163^(rd) embodiment, the disclosure provides a method of the161^(st) embodiment, which further comprises the step of purifying theexpressed protein.

In a 164^(th) embodiment, the disclosure provides a method of any one ofthe 151^(st) to 163^(rd) embodiments, wherein subjecting the recombinantautoactivated type I proelastase protein to autoactivation conditionscomprises raising the pH of a solution containing the recovered protein.

In a 165^(th) embodiment, the disclosure provides a method of the164^(th) embodiment, wherein the pH of the solution is raised to a basicpH.

In a 166^(th) embodiment, the disclosure provides a method of the165^(th) embodiment, wherein the basic pH is in a range from 7 to 9.

In a 167^(th) embodiment, the disclosure provides a method of the166^(th) embodiment, wherein the basic pH is 8.

In a 168^(th) embodiment, the disclosure provides a method of any one ofthe 164^(th) to 167^(th) embodiments, wherein the recovered protein isat a concentration of 10 mg/ml or less in said solution.

In a 169^(th) embodiment, the disclosure provides a method of any one ofthe 164^(th) to 167^(th) embodiments, wherein the recovered protein isat a concentration of 5 mg/ml or less in said solution.

In a 170^(th) embodiment, the disclosure provides a method of any one ofthe 164^(th) to 167^(th) embodiments, wherein the recovered protein isat a concentration of 2 mg/ml or less in said solution.

In a 171^(st) embodiment, the disclosure provides a method of any one ofthe 164^(th) to 167^(th) embodiments, wherein the recovered protein isat a concentration of 1 mg/ml or less in said solution.

In a 172^(nd) embodiment, the disclosure provides a method of any one ofthe 164^(th) to 167^(th) embodiments, wherein the recovered protein isat a concentration of 0.5 mg/ml or less in said solution.

In a 173^(rd) embodiment, the disclosure provides a method of any one ofthe 164^(th) to 167^(th) embodiments, wherein the recovered protein isat a concentration of 0.25 mg/ml or less in said solution.

In a 174^(th) embodiment, the disclosure provides a method of any one ofthe 165^(th) to 173^(rd) embodiments, wherein the recovered protein isat a concentration of at least 0.1 mg/ml in said solution.

In a 175^(th) embodiment, the disclosure provides a method of any one ofthe 165^(th) to 173^(rd) embodiments, wherein the recovered protein isat a concentration of at least 0.2 mg/ml in said solution.

In a 176^(th) embodiment, the disclosure provides a method of any one ofthe 165^(th) to 175^(th) embodiments wherein the recovered protein isexposed to the basic pH for a period of 0.5 to 8 hours.

In a 177^(th) embodiment, the disclosure provides a method of the174^(th) embodiment, wherein the recovered protein is exposed to thebasic pH for a period of 2 to 7 hours.

In a 178^(th) embodiment, the disclosure provides a method of the177^(th) embodiment, wherein the recovered protein is exposed to thebasic pH for a period of 6 hours.

In a 179^(th) embodiment, the disclosure provides a method of any one ofthe 165^(th) to 178^(th) embodiments, wherein said exposure to a basicpH is performed at a temperature of 22° C. to 28° C.

In a 180^(th) embodiment, the disclosure provides a method of the179^(th) embodiment, wherein said exposure to a basic pH is performed ata temperature of 26° C.

In a 181^(st) embodiment, the disclosure provides a method of the152^(nd) to 180^(th) embodiment, wherein the recovered protein is storedprior to autoactivation.

In a 182^(nd) embodiment, the disclosure provides a method of the181^(st) embodiment, wherein the recovered protein is lyophilized priorto storage.

In a 183^(rd) embodiment, the disclosure provides a method of the182^(nd) embodiment, wherein the recovered protein is purified prior tolyophilization.

In a 184^(th) embodiment, the disclosure provides a method of any one ofthe 181^(st) to 183^(rd) embodiments, wherein the recovered protein isstored for a period of at least one day, at least one week, at least onemonth or at least three months.

In a 185^(th) embodiment, the disclosure provides a method of the184^(th) embodiment, wherein the recovered protein is stored at atemperature from 80° C. to +4° C.

In a 186^(th) embodiment, the disclosure provides a method of any one ofthe 150^(th) to 185^(th) embodiments inasfar as such embodiments do notdepend on the 160^(th) or 162^(nd) embodiments, wherein the recombinantautoactivated type I proelastase is purified prior to subjecting it toautoactivation conditions.

In a 187^(th) embodiment, the disclosure provides a method of any one ofthe 150^(th) to 186^(th) embodiments, which further comprises the stepof isolating said mature type I elastase protein.

In a 188^(th) embodiment, the disclosure provides a method of any one ofthe 150^(th) to 187^(th) embodiments, wherein the mature type I elastaseprotein is a human type I mature elastase protein.

In a 189^(th) embodiment, the disclosure provides a method of any one ofthe 150^(th) to 187^(th) embodiments, wherein the mature type I elastaseprotein is a porcine type I mature elastase protein.

In a 190^(th) embodiment, the disclosure provides a method of producinga mature type I elastase protein comprising:

-   -   (a) culturing the host cell of the 84^(th) or 86^(th) embodiment        under conditions in which the protein is expressed;    -   (b) recovering the expressed protein;    -   (c) purifying the recovered protein;    -   (d) raising the pH of a solution containing the protein or        contacting the recovered protein with a catalytic amount of        elastase to produce a mature type I elastase protein,    -   thereby producing a mature type I elastase protein.

In a 191^(st) embodiment, the disclosure provides a method of the190^(th) embodiment, wherein the host cell is cultured in complexmedium.

In a 192^(nd) embodiment, the disclosure provides a method of the191^(st) embodiment, wherein the complex medium is bufferedmethanol-complex medium or buffered glycerol-complex medium.

In a 193^(rd) embodiment, the disclosure provides a method of any one ofthe 190^(th) to 192^(nd) embodiments, wherein the host cell is culturedin the presence of a citrate, succinate or acetate compound.

In a 194^(th) embodiment, the disclosure provides a method of the193^(rd) embodiment, wherein the citrate, succinate or acetate compoundis sodium citrate, sodium succinate or sodium acetate, respectively.

In a 195^(th) embodiment, the disclosure provides a method of the193^(rd) embodiment or 194^(th) embodiment, wherein one citrate,succinate or acetate compound is present in said culture at aconcentration of 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM,75-125 mM, or 90-110 mM.

In a 196^(th) embodiment, the disclosure provides a method of the193^(rd) embodiment or 194^(th) embodiment, wherein more than onecitrate, succinate or acetate compound is present in said culture, andwherein the total concentration of citrate, succinate or acetatecompounds in said solution is 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM,100-150 mM, 75-125 mM, or 90-110 mM.

In a 197^(th) embodiment, the disclosure provides a method of any one ofthe 190^(th) to 196^(th) embodiments, which further comprises the stepof (e) purifying said mature type I elastase.

In a 198^(th) embodiment, the disclosure provides a method of any one ofthe 190^(th) to 197^(th) embodiments, wherein the mature type I elastaseprotein is a human type I mature elastase protein.

In a 199^(th) embodiment, the disclosure provides a method of any one ofthe 190^(th) to 197^(th) embodiments, wherein the mature type I elastaseprotein is a porcine type I mature elastase protein.

In a 200^(th) embodiment, the disclosure provides a method of producinga mature type I elastase protein comprising:

-   -   (a) culturing the host cell of the 84^(th) or 86^(th) embodiment        under conditions in which the protein is expressed;    -   (b) recovering the expressed protein; and    -   (c) exposing the recovered protein to a basic pH until a mature        protein is produced;

thereby producing a mature type I elastase protein.

In a 201^(st) embodiment, the disclosure provides a method of the200^(th) embodiment, wherein the host cell is cultured in complexmedium.

In a 202^(nd) embodiment, the disclosure provides a method of the201^(st) embodiment, wherein the complex medium is bufferedmethanol-complex medium or buffered glycerol-complex medium.

In a 203^(rd) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 202^(nd) embodiments, wherein the host cell is culturedin the presence of a citrate, succinate or acetate compound.

In a 204^(th) embodiment, the disclosure provides a method of the203^(rd) embodiment, wherein the citrate, succinate or acetate compoundis sodium citrate, sodium succinate or sodium acetate, respectively.

In a 205^(th) embodiment, the disclosure provides a method of the203^(rd) embodiment or 204^(th) embodiment, wherein one citrate,succinate or acetate compound is present in said culture at aconcentration of 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM, 100-150 mM,75-125 mM, or 90-110 mM.

In a 206^(th) embodiment, the disclosure provides a method of the203^(rd) embodiment or the 204^(th) embodiment, wherein more than onecitrate, succinate or acetate compound is present in said culture, andwherein the total concentration of citrate, succinate or acetatecompounds in said solution is 5-50 mM, 7.5-100 mM, 10-150 mM, 50-200 mM,100-150 mM, 75-125 mM, or 90-110 mM.

In a 207^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 206^(th) embodiments, wherein the basic pH is 7 to 9.

In a 208^(th) embodiment, the disclosure provides a method of the207^(th) embodiment, wherein the basic pH is 8.

In a 209^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 208^(th) embodiments, wherein recovered protein is at aconcentration of 10 mg/ml or less when exposed to the basic pH.

In a 210^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 208^(th) embodiments, wherein recovered protein is at aconcentration of 5 mg/ml or less when exposed to the basic pH.

In a 211^(st) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 208^(th) embodiments, wherein recovered protein is at aconcentration of 2 mg/ml or less when exposed to the basic pH.

In a 212^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 208^(th) embodiments, wherein recovered protein is at aconcentration of 1 mg/ml or less when exposed to the basic pH.

In a 213^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 208^(th) embodiments, wherein recovered protein is at aconcentration of 0.5 mg/ml or less when exposed to the basic pH.

In a 214^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 208^(th) embodiments, wherein recovered protein is at aconcentration of 0.25 mg/ml or less when exposed to the basic pH.

In a 215^(th) embodiment, the disclosure provides a method of any one ofthe 209^(th) to 214^(th) embodiments, wherein recovered protein is at aconcentration of at least 0.1 mg/ml when exposed to the basic pH.

In a 216^(th) embodiment, the disclosure provides a method of any one ofthe 209^(th) to 214^(th) embodiments, wherein recovered protein is at aconcentration of at least 0.2 mg/ml when exposed to the basic pH.

In a 217^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 214^(th) embodiments, wherein the recovered protein isexposed to the basic pH for a period of 0.5 to 8 hours.

In a 218^(th) embodiment, the disclosure provides a method of the217^(th) embodiment, wherein the recovered protein is exposed to thebasic pH for a period of 2 to 7 hours.

In a 219^(th) embodiment, the disclosure provides a method of the218^(th) embodiment, wherein the recovered protein is exposed to thebasic pH for a period of 6 hours.

In a 220^(th) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 219^(th) embodiments, wherein said exposure to a basicpH is performed at a temperature of 22° C. to 28° C.

In a 221^(st) embodiment, the disclosure provides a method of the220^(th) embodiment, wherein said exposure to a basic pH is performed ata temperature of 26° C.

In a 222^(nd) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 221^(st) embodiments, which further comprises the stepof (d) isolating said mature type I elastase protein.

In a 223^(rd) embodiment, the disclosure provides a method of any one ofthe 150^(th) to 222^(nd) embodiments, wherein the mature type I elastaseis a mature porcine type I elastase.

In a 224^(th) embodiment, the disclosure provides a method of any one ofthe 150^(th) to 222^(nd) embodiments, wherein the mature type I elastaseis a mature human type I elastase.

In a 225^(th) embodiment, the disclosure provides a method of producinga formulation of mature elastase protein, comprising:

-   -   (a) subjecting an autoactivated proelastase protein to        autoactivating conditions to produce mature elastase protein;        and    -   (b) formulating the mature elastase protein,

thereby producing a formulation of mature elastase protein.

In a 226^(th) embodiment, the disclosure provides a method of the225^(th) embodiment, wherein the autoactivated proelastase protein isrecombinant.

In a 227^(th) embodiment, the disclosure provides a method of the226^(th) embodiment further comprising, prior to step (a) recovering theproelastase protein from a culture of a host cell capable of expressingsaid autoactivated proelastase protein grown under conditions in whichthe proelastase protein is expressed.

In a 228^(th) embodiment, the disclosure provides a method of the225^(th) embodiment or the 226^(th) embodiment, further comprising,prior to step (b), purifying said mature elastase protein.

In a 229^(th) embodiment, the disclosure provides a method of any one ofthe 225^(th) to 228^(th) embodiments, wherein said formulating stepcomprises lyophilizing said mature elastase protein.

In a 230^(th) embodiment, the disclosure provides a method of the229^(th) embodiment, wherein the mature elastase protein is not mixedwith buffer or buffer ingredients prior to lyophilization.

In a 231^(st) embodiment, the disclosure provides a method of the230^(th) embodiment, wherein the mature elastase protein is mixed withone or more buffer ingredients following lyophilization.

In a 232^(nd) embodiment, the disclosure provides a method of the229^(th) embodiment, wherein the mature elastase protein is mixed withbuffer or one or more buffer ingredients prior to lyophilization.

In a 233^(rd) embodiment, the disclosure provides a method of the231^(st) or 232^(nd) embodiment, wherein the buffer is a phosphatebuffered saline (“PBS”) buffer or the buffer ingredients are PBS bufferingredients.

In a 234^(th) embodiment, the disclosure provides a method of any one ofthe 231^(st) to 233^(rd) embodiments, wherein the buffer comprisesdextran or wherein the buffer ingredients comprise dextran.

In a 235^(th) embodiment, the disclosure provides a method of the234^(th) embodiment, wherein the dextran is dextran-18.

In a 236^(th) embodiment, the disclosure provides a method of any one ofthe 230^(th) to 235^(th) embodiments, wherein the buffer comprisespolysorbate 80.

In a 237^(th) embodiment, the disclosure provides a method of any one ofthe 229^(th) to 236^(th) embodiments, wherein said formulating stepfurther comprises reconstituting the lyophilized mature elastase proteinwith a liquid.

In a 238^(th) embodiment, the disclosure provides a method of the237^(th) embodiment, wherein the liquid is water.

In a 239^(th) embodiment, the disclosure provides a method of the237^(th) embodiment, wherein the liquid is a buffer.

In a 240^(th) embodiment, the disclosure provides a method of the239^(th) embodiment, wherein the buffer is full strength buffer, greaterthan full strength buffer, or less than full strength buffer.

In a 241^(st) embodiment, the disclosure provides a method of any one ofthe 237^(th) to 240^(th) embodiments, wherein upon reconstitution asolution of mature elastase protein in full strength buffer, greaterthan full strength buffer, or less than full strength buffer isproduced.

In a 242^(nd) embodiment, the disclosure provides a method of the241^(st) embodiment, wherein buffer ingredients are present in thelyophilisate, added upon reconstitution, or both.

In a 243^(rd) embodiment, the disclosure provides a method of any one ofthe 240^(th) to 242^(nd) embodiments, wherein full strength buffercomprises 1×PBS.

In a 244^(th) embodiment, the disclosure provides a method of the240^(th) or 241^(st) embodiment, wherein the less than full strengthbuffer comprises 0.1×PBS to 0.5×PBS.

In a 245^(th) embodiment, the disclosure provides a method of the244^(th) embodiment, wherein the less than full strength buffercomprises 0.1×PBS.

In a 246^(th) embodiment, the disclosure provides a method of the244^(th) embodiment, wherein the less than full strength buffercomprises 0.5×PBS.

In a 247^(th) embodiment, the disclosure provides a method of the240^(th) or 241^(st) embodiment, wherein the greater than full strengthbuffer comprises 1.1×PBS to 3×PBS.

In a 248^(th) embodiment, the disclosure provides a method of the247^(th) embodiment, wherein the greater than full strength buffercomprises 1.5×PBS to 2×PBS.

In a 249^(th) embodiment, the disclosure provides a method of any one ofthe 241^(st) to 248^(th) embodiments, wherein the buffer comprisesdextran.

In a 250^(th) embodiment, the disclosure provides a method of the249^(th) embodiment, wherein the dextran is dextran 18.

In a 251^(st) embodiment, the disclosure provides a method of any one ofthe 241^(st) to 250^(th) embodiments, wherein the buffer comprisespolysorbate 80.

In a 252^(nd) embodiment, the disclosure provides a method of any one ofthe 241^(st) to 251^(st) embodiments, wherein the buffer is at a pH of 7to 8.

In a 253^(rd) embodiment, the disclosure provides a method of the252^(nd) embodiment, wherein the buffer is at a pH of 7.4.

In a 254^(th) embodiment, the disclosure provides a method of any one ofthe 237^(th) to 253^(rd) embodiments, wherein the mature elastaseprotein is reconstituted to a concentration of 0.001 mg/ml to 50 mg/ml.

In a 255^(th) embodiment, the disclosure provides a method of the254^(th) embodiment, wherein the mature elastase protein isreconstituted to a concentration of 0.1 mg/ml to 40 mg/ml.

In a 256^(th) embodiment, the disclosure provides a method of the255^(th) embodiment, wherein the mature elastase protein isreconstituted to a concentration of 5 mg/ml to 30 mg/ml.

In a 257^(th) embodiment, the disclosure provides a method of the256^(th) embodiment, wherein the mature elastase protein isreconstituted to a concentration of 10 mg/ml to 20 mg/ml.

In a 258^(th) embodiment, the disclosure provides a method of any one ofthe 225^(th) to 257^(th) embodiments, wherein said formulation is apharmaceutical composition comprising said mature elastase protein.

In a 259^(th) embodiment, the disclosure provides a method of any one ofthe 225^(th) to 258^(th) embodiments, wherein the mature type I elastaseprotein is a human type I mature elastase protein.

In a 260^(th) embodiment, the disclosure provides a method of any one ofthe 225^(th) to 258^(th) embodiments, wherein the mature type I elastaseprotein is a porcine type I mature elastase protein.

In a 261^(st) embodiment, the disclosure provides a method of producinga lyophilized mature type I elastase comprising:

-   -   (a) producing a mature type I elastase according to the method        of any one of the 151^(st) to 181^(st) embodiments;    -   (b) isolating mature type I elastase; and    -   (c) lyophilizing said isolated mature type I elastase,

thereby producing a lyophilized mature type I elastase.

In a 262^(nd) embodiment, the disclosure provides a method of the261^(st) embodiment, wherein the lyophilized mature type I elastase is95% to 100% pure.

In a 263^(rd) embodiment, the disclosure provides a method of the261^(st) embodiment or 262^(nd) embodiment, wherein the lyophilizedmature type I elastase is at least 95% pure.

In a 264^(th) embodiment, the disclosure provides a method of the261^(st) embodiment or 262^(nd) embodiment, wherein the lyophilizedmature type I elastase is at least 98% pure.

In a 265^(th) embodiment, the disclosure provides a method of any one ofthe 261^(st) to 264^(th) embodiments, wherein the lyophilized maturetype I elastase is purified to homogeneity.

In a 266^(th) embodiment, the disclosure provides a method of any one ofthe 261^(st) to 265^(th) embodiments, wherein the mature type I elastaseprotein is a human type I mature elastase protein.

In a 267^(th) embodiment, the disclosure provides a method of any one ofthe 261^(st) to 265^(th) embodiments, wherein the mature type I elastaseprotein is a porcine type I mature elastase protein.

In a 268^(th) embodiment, the disclosure provides a method of any one ofthe 130^(th), 149^(th), 188^(th), 198^(th), 223^(rd), 259^(th), and267^(th) embodiments, wherein the mature human type I elastase proteinconsists essentially of SEQ ID NO:5, SEQ ID NO:84, or SEQ ID NO:87.

In a 269^(th) embodiment, the disclosure provides a method of the268^(th) embodiment, wherein the mature human type I elastase proteinconsists essentially of SEQ ID NO:1 or SEQ ID NO:4.

In a 270^(th) embodiment, the disclosure provides a method of any one ofthe 131^(st), 150^(th), 189^(th), 199^(th), 224^(th), 260^(th), and268^(th) embodiments, wherein the mature porcine type I elastase proteinconsists essentially of SEQ ID NO:39.

In a 271^(st) embodiment, the disclosure provides a method of any one ofthe 111^(st) to 128^(th), 132^(nd) to 148^(th), 151^(st) to 197^(th) and225^(th) to 257^(th) embodiments, wherein the proelastase protein is aprotein of any one of embodiments 13 to 27.

In a 272^(nd) embodiment, the disclosure provides a method of any one ofthe 200^(th) to 222^(nd) embodiments, wherein the expressed protein is aprotein of any one of embodiments 13 to 27.

In a 273^(rd) embodiment, the disclosure provides a mature human type Ielastase produced by or obtainable by the method of any one of the149^(th), 188^(th), 198^(th) and 223^(rd) embodiments.

In a 274^(th) embodiment, the disclosure provides a mature human type Ielastase of the 273^(rd) embodiment which has a specific activity of 1to 40 U/mg protein.

In a 275^(th) embodiment, the disclosure provides a mature porcine typeI elastase produced by or obtainable by the method of any one of the150^(th), 189^(th), 199^(th) and 224^(th) embodiments.

In a 276^(th) embodiment, the disclosure provides a mature human type Ielastase of the 275^(th) embodiment which has a specific activity of 10to 100 U/mg protein.

In a 277^(th) embodiment, the disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of (a) themature human type I elastase of the 273^(rd) embodiment or 274^(th)embodiment or (b) a formulation of mature human type I elastase producedby or obtainable by the method of the 259^(th) embodiment.

In a 278^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 277^(th) embodiment, wherein the mature human type Ielastase protein consists essentially of SEQ ID NO:5, SEQ ID NO:84, orSEQ ID NO:87.

In a 279^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 278^(th) embodiment, wherein the mature human type Ielastase protein consists essentially of SEQ ID NO:1 or SEQ ID NO:4.

In a 280^(th) embodiment, the disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of (a) themature porcine type I elastase of the 275^(th) embodiment or 276^(th)embodiment or (b) a formulation of mature porcine type I elastaseproduced by or obtainable by the method of the 260^(th) embodiment.

In a 281^(st) embodiment, the disclosure provides a pharmaceuticalcomposition of the 280^(th) embodiment, wherein the mature porcine typeI elastase protein consists essentially of SEQ ID NO:39.

In a 282^(nd) embodiment, the disclosure provides a pharmaceuticalcomposition comprising (i) a therapeutically effective amount of maturehuman type I elastase and (ii) a pharmaceutically acceptable carrier,which pharmaceutical composition is characterized by at least one of thefollowing properties:

-   -   (a) the composition is free of trypsin;    -   (b) the composition is substantially free of trypsin;    -   (c) the composition is free of any protein consisting of SEQ ID        NOS: 2, 3, 37, 38, 70 and/or 71;    -   (d) the composition is substantially free of any protein        consisting of SEQ ID NOS: 2, 3, 37, 38, 70 and/or 71;    -   (e) the composition is free of bacterial proteins;    -   (f) the composition is substantially free of bacterial proteins;    -   (g) the composition is free of mammalian proteins other than        said mature human type I elastase;    -   (h) the composition is substantially free of mammalian proteins        other than said mature human type I elastase.

In a 283^(rd) embodiment, the disclosure provides a pharmaceuticalcomposition comprising (i) a therapeutically effective amount of maturehuman type I elastase and (ii) a pharmaceutically acceptable carrier,which pharmaceutical composition is characterized by at least one of thefollowing properties:

-   -   (a) the composition is free of trypsin;    -   (b) the composition is substantially free of trypsin;    -   (c) the composition is free of any protein consisting of SEQ ID        NOS:70 and 71;    -   (d) the composition is substantially free of any protein        consisting of SEQ ID NOS:2 and 3;    -   (e) the composition is free of bacterial proteins;    -   (f) the composition is substantially free of bacterial proteins;    -   (g) the composition is free of mammalian proteins other than        said mature human type I elastase;    -   (h) the composition is substantially free of mammalian proteins        other than said mature human type I elastase.

In a 284^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 282^(nd) embodiment or 283^(rd) embodiment which ischaracterized by at least two of the properties (a) to (h).

In a 285^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 284^(th) embodiment, wherein said at least twoproperties include (a) and (c).

In a 286^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 284^(th) embodiment, wherein said at least twoproperties include (b) and (d).

In a 287^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 282^(nd) embodiment or 283^(rd) embodiment which ischaracterized by at least three of the properties (a) to (h).

In a 288^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 284^(th) embodiment, wherein said at least threeproperties include (a), (c) and (e).

In a 289^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 284^(th) embodiment, wherein said at least threeproperties include (b), (d), and (f).

In a 290^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 282^(nd) embodiment or 283^(rd) embodiment which ischaracterized by at least four of the properties (a) to (h).

In a 291^(st) embodiment, the disclosure provides a pharmaceuticalcomposition of the 284^(th) embodiment, wherein said at least fourproperties include (a), (c), (e) and (g).

In a 292^(nd) embodiment, the disclosure provides a pharmaceuticalcomposition of the 284^(th) embodiment, wherein said at least fourproperties include (b), (d), (f), and (h).

In a 293^(rd) embodiment, the disclosure provides a pharmaceuticalcomposition of the 282^(nd) embodiment or 283^(rd) embodiment which ischaracterized by at least five of the properties (a) to (h).

In a 294^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 282^(nd) embodiment or 283^(rd) embodiment which ischaracterized by at least six of the properties (a) to (h).

In a 295^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 282^(nd) embodiment or 283^(rd) embodiment which ischaracterized by at least seven of the properties (a) to (h).

In a 296^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 282^(nd) embodiment or 283^(rd) embodiment which ischaracterized by all properties (a) to (h).

In a 297^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 296^(th) embodiments which isfree or substantially free of one, two, three or all four proteinsconsisting of SEQ ID NO:85, 86, 94 and 95.

In a 298^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 297^(th) embodiments which isfree or substantially free of proteins consisting of SEQ ID NO:85 andSEQ ID NO:86.

In a 299^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 297^(th) embodiments which isfree or substantially free of proteins consisting of SEQ ID NO:94 andSEQ ID NO:95.

In a 300^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 297^(th) embodiments which isfree or substantially free of one, two, or all three proteins consistingof SEQ ID NO:106, 107 and 108.

In a 301^(st) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 300^(th) embodiments whichcontains pharmaceutically acceptable levels of endotoxins.

In a 302^(nd) embodiment, the disclosure provides a pharmaceuticalcomposition of the 301^(st) embodiment, wherein said pharmaceuticalcomposition is a liquid composition and wherein said pharmaceuticallyacceptable levels of endotoxins are 8 EU/ml or less.

In a 303^(rd) embodiment, the disclosure provides a pharmaceuticalcomposition of the 302^(nd) embodiment, wherein said pharmaceuticallyacceptable levels of endotoxins are 5 EU/ml or less.

In a 304^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 301^(st) embodiment, wherein said pharmaceuticalcomposition is a solid composition and wherein said pharmaceuticallyacceptable levels of endotoxins are 10 EU or less per gram of maturehuman type I elastase.

In a 305^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 304^(th) embodiment wherein said pharmaceuticallyacceptable levels of endotoxins are 5 EU or less per gram of maturehuman type I elastase.

In a 306^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 305^(th) embodiments in whichthe mature human type I elastase is characterized by a specific activityof 1 to 40 U/mg of protein.

In a 307^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 306^(th) embodiment in which the mature human type Ielastase is characterized by a specific activity of 25 to 35 U/mg ofprotein.

In a 308^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 306^(th) embodiment in which the mature human type Ielastase is characterized by a specific activity of greater than 10 U/mgof protein.

In a 309^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 306^(th) embodiment in which the mature human type Ielastase is characterized by a specific activity of greater than 20 U/mgof protein.

In a 310^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 309^(th) embodiments, whereinthe mature human type I elastase consists essentially of SEQ ID NO: 5,84, or 87.

In a 311^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 310^(th) embodiment, wherein the mature human type Ielastase consists essentially of SEQ ID NO:1 or SEQ ID NO:4.

In a 312^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of any one of the 282^(nd) to 311^(th) embodiments in whichthe trypsin activity corresponds to less than 4 ng per 1 mg of maturehuman type I elastase protein.

In a 313^(th) embodiment, the disclosure provides a pharmaceuticalcomposition of the 312^(th) embodiment in which the trypsin activitycorresponds to less than 2 ng per 1 mg of mature human type I elastaseprotein.

In a 314^(th) embodiment, the disclosure provides a lyophilizedformulation of a protein consisting essentially of SEQ ID NO: 5, 84, or87 which upon reconstitution to a concentration of said protein of 1mg/ml the trypsin activity corresponds to less than 2 ng/ml trypsin.

In a 315^(th) embodiment, the disclosure provides a lyophilizedformulation of the 314^(th) embodiment, wherein the moisture content isless than 5%.

In a 316^(th) embodiment, the disclosure provides a lyophilizedformulation of the 315^(th) embodiment which comprises sodium ions,potassium ions, phosphate ions, and chloride ions.

In a 317^(th) embodiment, the disclosure provides a lyophilizedformulation of the 315^(th) embodiment which comprises polysorbate-80.

In a 318^(th) embodiment, the disclosure provides a lyophilizedformulation of the 315^(th) embodiment which comprises dextran.

In a 319^(th) embodiment, the disclosure provides a lyophilizedformulation of the 318^(th) embodiment, wherein the dextran isdextran-18.

In a 320^(th) embodiment, the disclosure provides a lyophilizedformulation of the 311^(th) embodiment which comprises sodium ions,potassium ions, phosphate ions, chloride ions and polysorbate-80.

In a 321^(st) embodiment, the disclosure provides a lyophilizedformulation of the 314^(th) embodiment which comprises sodium ions,potassium ions, phosphate ions, chloride ions and dextran.

In a 322^(nd) embodiment, the disclosure provides a lyophilizedformulation of the 321^(st) embodiment, wherein the dextran isdextran-18.

In a 323^(rd) embodiment, the disclosure provides a lyophilizedformulation of the 314^(th) embodiment which comprises sodium ions,potassium ions, phosphate ions, chloride ions, polysorbate-80, anddextran.

In a 324^(th) embodiment, the disclosure provides a lyophilizedformulation of the 323^(rd) embodiment, wherein the dextran isdextran-18.

In a 325^(th) embodiment, the disclosure provides a liquid formulationcomprising a solution of a least 0.1 mg/ml of a protein consistingessentially of SEQ ID NO: 5, 84, or 87 in which the trypsin activitycorresponds to less than 2 ng/ml trypsin.

In a 326^(th) embodiment, the disclosure provides a liquid formulationof the 325^(th) embodiment, wherein the solution is a buffered solution.

In a 327^(th) embodiment, the disclosure provides a liquid formulationof the 326^(th) embodiment, wherein the solution is buffered to a pH of7 to 8.

In a 328^(th) embodiment, the disclosure provides a liquid formulationof the 326^(th) embodiment in which the solution is PBS.

In a 329^(th) embodiment, the disclosure provides a liquid formulationof the 328^(th) embodiment in which the solution comprises 137 mM sodiumchloride, 2.7 mM potassium phosphate, and 10 mM sodium phosphate.

In a 330^(th) embodiment, the disclosure provides a liquid formulationof any one of the 325^(th) to 329^(th) embodiments which furthercomprises one or more excipients.

In a 331^(st) embodiment, the disclosure provides a liquid formulationof the 330^(th) embodiment in which said one or more excipientscomprises dextran-18.

In a 332^(nd) embodiment, the disclosure provides a liquid formulationof the 331^(st) embodiment in which the dextran-18 is in the amount of8% weight/volume of said solution.

In a 333^(rd) embodiment, the disclosure provides a liquid formulationof the 330^(th) embodiment in which said one or more excipientscomprises polysorbate-80.

In a 334^(th) embodiment, the disclosure provides a liquid formulationof the 333^(rd) embodiment in which the polysorbate-80 is in the amountof 0.01% weight/volume of said solution.

In a 335^(th) embodiment, the disclosure provides a liquid formulationof any one of the 325^(th) to 334^(th) embodiments in which theconcentration of said protein in said solution is in the range of 0.001mg/ml to 40 mg/ml.

In a 336^(th) embodiment, the disclosure provides a liquid formulationof the 335^(th) embodiment in which the concentration of said protein insaid solution is in the range of 0.1 mg/ml to 20 mg/ml.

In a 337^(th) embodiment, the disclosure provides a liquid formulationof any one of the 325^(th) to 336^(th) embodiments with furthercomprises one or more of a preservative, a solubilizer and a coloringagent.

In a 338^(th) embodiment, the disclosure provides a liquid formulationof any one of the 325^(th) to 337^(th) embodiments which issubstantially free of mammalian proteins other than said protein of SEQID NO:5, 84 or 87.

In a 339^(th) embodiment, the disclosure provides a liquid formulationof any one of the 325^(th) to 338^(th) embodiments which issubstantially free of bacterial proteins.

In a 340^(th) embodiment, the disclosure provides a formulation made byor obtainable by the method of any one of the 225^(th) to 258^(th)embodiments.

In a 341^(st) embodiment, the disclosure provides a formulation of the340^(th) embodiment which is a liquid formulation.

In a 342^(nd) embodiment, the disclosure provides a formulation of the340^(th) or 341^(st) embodiment in which the mature type I elastaseprotein is a human type I mature elastase protein.

In a 343^(rd) embodiment, the disclosure provides a formulation of the342^(nd) embodiment, wherein the mature human type I elastase proteinconsists essentially of SEQ ID NO:5, SEQ ID NO:84, or SEQ ID NO:87.

In a 344^(th) embodiment, the disclosure provides a formulation of the342^(nd) embodiment, wherein the mature human type I elastase proteinconsists essentially of SEQ ID NO:1 or SEQ ID NO:4.

In a 345^(th) embodiment, the disclosure provides a formulation of the340^(th) or 341^(st) embodiment in which the mature type I elastaseprotein is a porcine type I mature elastase protein.

In a 346^(th) embodiment, the disclosure provides a formulation of the345^(th) embodiment, wherein the mature porcine type I elastase proteinconsists essentially of SEQ ID NO:39.

In a 347^(th) embodiment, the disclosure provides a method of removingone or more incorrectly processed mature elastase proteins from amixture of correctly and incorrectly processed mature elastase proteins,said method comprising:

-   -   (a) subjecting a composition comprising a mixture of correctly        and incorrectly processed mature elastase proteins to a pH at        which the correctly processed mature enzyme is active;    -   (b) maintaining such pH until such time that one or more        incorrectly processed mature elastase proteins are degraded,

thereby removing said one or more incorrectly processed mature elastaseproteins from a mixture of correctly and incorrectly processed matureelastase proteins.

In a 348^(th) embodiment, the disclosure provides a method of the347^(th) embodiment, wherein said one or more incorrectly processedmature elastase proteins contain at least one additional or fewer aminoacid at the N-terminus relative to correctly processed mature elastaseproteins.

In a 349^(th) embodiment, the disclosure provides a method of the347^(th) or 348^(th) embodiment wherein said pH is between 5 and 12.

In a 350^(th) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 349^(th) embodiments, wherein said one or moreincorrectly processed mature elastase proteins are degraded by 50% to100%.

In a 351^(st) embodiment, the disclosure provides a method of the348^(th) embodiment, wherein said one or more incorrectly processedmature elastase proteins are degraded by 50% to 99%.

In a 352^(nd) embodiment, the disclosure provides a method of the348^(th) embodiment, wherein said one or more incorrectly processedmature elastase proteins are degraded by 50% to 98%.

In a 353^(rd) embodiment, the disclosure provides a method of the348^(th) embodiment, wherein said one or more incorrectly processedmature elastase proteins are degraded by 50% to 95%.

In a 354^(th) embodiment, the disclosure provides a method of the348^(th) embodiment, wherein said one or more incorrectly processedmature elastase proteins are degraded by 50% to 90%.

In a 355^(th) embodiment, the disclosure provides a method of any one ofthe 350^(th) to 354^(th) embodiments, wherein more than one incorrectlyprocessed mature elastase protein is degraded by at least 50%.

In a 356^(th) embodiment, the disclosure provides a method of the355^(th) embodiment, wherein all incorrectly processed mature elastaseproteins in the mixture are degraded by at least 50%.

In a 357^(th) embodiment, the disclosure provides a method of any one ofthe 350^(th) to 354^(th) embodiments wherein at least one incorrectlyprocessed mature elastase protein is degraded by at least 75%.

In a 358^(th) embodiment, the disclosure provides a method of the357^(th) embodiment, wherein more than one incorrectly processed matureelastase protein is degraded by at least 75%.

In a 359^(th) embodiment, the disclosure provides a method of the357^(th) embodiment, wherein all incorrectly processed mature elastaseproteins in the mixture are degraded by at least 75%.

In a 360^(th) embodiment, the disclosure provides a method of any one ofthe 350^(th) to 354^(th) embodiments wherein at least one incorrectlyprocessed mature elastase protein is degraded by at least 90%.

In a 361^(st) embodiment, the disclosure provides a method of the360^(th) embodiment, wherein more than one incorrectly processed matureelastase protein is degraded by at least 90%.

In a 362^(nd) embodiment, the disclosure provides a method of the360^(th) embodiment, wherein all incorrectly processed mature elastaseproteins in the mixture are degraded by at least 90%.

In a 363^(rd) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 362^(nd) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of 10 mg/ml or less.

In a 364^(th) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 362^(nd) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of 5 mg/ml or less.

In a 365^(th) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 362^(nd) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of 2 mg/ml or less.

In a 366^(th) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 362^(nd) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of 1 mg/ml or less.

In a 367^(th) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 362^(nd) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of 0.5 mg/ml or less.

In a 368^(th) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 362^(nd) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of 0.25 mg/ml or less.

In a 369^(th) embodiment, the disclosure provides a method of any one ofthe 363^(rd) to 368^(th) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of at least 0.1 mg/ml.

In a 370^(th) embodiment, the disclosure provides a method of any one ofthe 363^(rd) to 368^(th) embodiments, wherein the composition is aliquid composition in which the total elastase protein is at aconcentration of at least 0.2 mg/ml.

In a 371^(st) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 370^(th) embodiments, wherein the trypsin activity insaid composition is less than 4 ng/ml trypsin per mg of total elastaseproteins.

In a 372^(nd) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 370^(th) embodiments, wherein the trypsin activity insaid composition is less than 2 ng/ml trypsin per mg of total elastaseproteins.

In a 373^(rd) embodiment, the disclosure provides a method of any one ofthe 347^(th) to 372^(nd) embodiments, wherein the composition is free orsubstantially free of a protein consisting of SEQ ID NO:104 and/or isfree or substantially free of a protein consisting of SEQ ID NO:105.

In a 374^(th) embodiment, the disclosure provides a method of producinga pharmaceutical composition comprising a mature type I elastase, saidmethod comprising (i) producing a lyophilized mature type I elastase bythe method of any one of the 261^(st) to 265^(th) embodiments;

and (ii) reconstituting the lyophilized mature type I elastase in wateror a pharmaceutically acceptable carrier, thereby producing apharmaceutical composition comprising a mature human type I elastase.

In a 375^(th) embodiment, the disclosure provides a method of producinga pharmaceutical composition comprising a mature type I elastase, saidmethod comprising reconstituting the lyophilized formulation of any oneof the 314^(th) to 324^(th) embodiments in water or a pharmaceuticallyacceptable carrier, thereby producing a pharmaceutical compositioncomprising a mature type I elastase.

In a 376^(th) embodiment, the disclosure provides a method of the374^(th) embodiment or the 375^(th) embodiment where the pharmaceuticalcomposition comprises phosphate.

In a 377^(th) embodiment, the disclosure provides a method of any one ofthe 374^(th) to 376^(th) embodiments, wherein the mature type I elastaseis mature human type I elastase.

In a 378^(th) embodiment, the disclosure provides a method of the377^(th) embodiment characterized by a specific activity of 1 to 40 U/mgprotein.

In a 379^(th) embodiment, the disclosure provides a method of the377^(th) embodiment wherein the mature type I elastase is characterizedby a specific activity of 25 to 35 U/mg protein.

In a 380^(th) embodiment, the disclosure provides a method of any one ofthe 377^(th) to 379^(th) embodiments, wherein the mature human type Ielastase in said pharmaceutical composition maintains 60% to 100% of itsspecific activity after at least a week of storage at 4° C., after atleast a month of storage at 4° C., after at least two months of storageat 4° C., after at least three months of storage at 4° C., or after atleast month six months of storage at 4° C.

In a 381^(st) embodiment, the disclosure provides a method of any one ofthe 377^(th) to 379^(th) embodiments, wherein the mature human type Ielastase in said pharmaceutical composition maintains 60% to 98% of itsspecific activity after at least a week of storage at 4° C., after atleast a month of storage at 4° C., after at least two months of storageat 4° C., after at least three months of storage at 4° C., or after atleast month six months of storage at 4° C.

In a 382^(nd) embodiment, the disclosure provides a method of any one ofthe 377^(th) to 379^(th) embodiments, wherein the mature human type Ielastase in said pharmaceutical composition maintains 60% to 95% of itsspecific activity after at least a week of storage at 4° C., after atleast a month of storage at 4° C., after at least two months of storageat 4° C., after at least three months of storage at 4° C., or after atleast month six months of storage at 4° C.

In a 383^(rd) embodiment, the disclosure provides a method of any one ofthe 377^(th) to 379^(th) embodiments, wherein the mature human type Ielastase in said pharmaceutical composition maintains 60% to 90% of itsspecific activity after at least a week of storage at 4° C., after atleast a month of storage at 4° C., after at least two months of storageat 4° C., after at least three months of storage at 4° C., or after atleast month six months of storage at 4° C.

In a 384^(th) embodiment, the disclosure provides a method of any one ofthe 377^(th) to 379^(th) embodiments, wherein the mature human type Ielastase in said pharmaceutical composition maintains 60% to 80% of itsspecific activity after at least a week of storage at 4° C., after atleast a month of storage at 4° C., after at least two months of storageat 4° C., after at least three months of storage at 4° C., or after atleast month six months of storage at 4° C.

In a 385^(th) embodiment, the disclosure provides a method of any one ofthe 377^(th) to 384^(th) embodiments, wherein the mature human type Ielastase in said pharmaceutical composition maintains at least 70% ofits specific activity after a week of storage at 4° C.

In a 386^(th) embodiment, the disclosure provides a pharmaceuticalcomposition produced by or obtainable by the method of any one of the374^(th) to 385^(th) embodiments.

In a 387^(th) embodiment, the disclosure provides a method fortherapeutically increasing the diameter of an artery or vein in a humansubject in need thereof, the method comprising: locally administering tothe wall of the artery or vein in the human subject (a) thepharmaceutical composition of any one of the 277^(th) to 313^(th) and386^(th) embodiments, (b) the liquid formulation of any one of the325^(th) to 339^(th) embodiments, or (c) the formulation of the 341^(st)embodiment in a dose sufficient to increase the diameter of the arteryor vein.

In a 388^(th) embodiment, the disclosure provides a method of the387^(th) embodiment, wherein the diameter of the vessel, the lumenaldiameter of the vessel, or both, are increased.

In a 389^(th) embodiment, the disclosure provides a method forpreventing or treating vasospasm of an artery or vein in a human subjectin need thereof, the method comprising:

locally administering to the wall of the artery or vein in the humansubject (a) the pharmaceutical composition of any one of the 277^(th) to313^(th) and 386^(th) embodiments, (b) the liquid formulation of any oneof the 325^(th) to 339^(th) embodiments, or (c) the formulation of the341^(st) embodiment in a dose sufficient to prevent or treat vasospasmof the artery or vein.

In a 390^(th) embodiment, the disclosure provides a method for treatingan obstructed artery or vein in a human subject in need of suchtreatment, the method comprising: locally administering to the wall ofthe artery or vein in the human subject (a) the pharmaceuticalcomposition of any one of the 277^(th) to 313^(th) and 386^(th)embodiments, (b) the liquid formulation of any one of the 325^(th) to339^(th) embodiments, or (c) the formulation of the 341^(st) embodiment,wherein said administration results in proteolysis of elastin in thewall of the artery or vein leading to enlargement of the diameter of theartery or vein.

In a 391^(st) embodiment, the disclosure provides a method for treatingan artery or vein connected to an arteriovenous hemodialysis graft orarteriovenous fistula in a human subject in need of such treatment, themethod comprising: locally administering to the wall of the artery orvein in the human subject ((a) the pharmaceutical composition of any oneof the 277^(th) to 313^(th) and 386^(th) embodiments, (b) the liquidformulation of any one of the 325^(th) to 339^(th) embodiments, or (c)the formulation of the 341^(st) embodiment, wherein said administrationresults in proteolysis of elastin in the wall of the artery or veinleading to enlargement of the diameter of the artery or vein.

In a 392^(nd) embodiment, the disclosure provides a method for treatinga vein in a human subject for use in hemodialysis, the methodcomprising: locally administering to the wall of the vein in the humansubject (a) the pharmaceutical composition of any one of the 277^(th) to313^(th) and 386^(th) embodiments, (b) the liquid formulation of any oneof the 325^(th) to 339^(th) embodiments, or (c) the formulation of the341^(st) embodiment, wherein said administration results in proteolysisof elastin in the wall of the vein leading to enlargement of thediameter of the vein.

In a 393^(rd) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 392^(nd) embodiments, which further comprises insertinga portion of a delivery apparatus into the wall of the artery or vein todeliver elastase to the wall of the artery or vein.

In a 394^(th) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 393^(rd) embodiments, wherein the pharmaceuticalcomposition or liquid formulation is administered by a catheter.

In a 395^(th) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 394^(th) embodiments, wherein the wherein thepharmaceutical composition or liquid formulation is administereddirectly into the wall of the artery or vein.

In a 396^(th) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 395^(th) embodiments, wherein the artery or vein isobstructed.

In a 397^(th) embodiment, the disclosure provides a method of the396^(th) embodiment, wherein the artery or vein is obstructed bystenosis.

In a 398^(th) embodiment, the disclosure provides a method of the397^(th) embodiment, wherein the obstruction permits passage of aninsufficient volume of blood prior to the treatment.

In a 399^(th) embodiment, the disclosure provides a method of the398^(th) embodiment, wherein the obstruction is a stenosis.

In a 400^(th) embodiment, the disclosure provides a method of the399^(th) embodiment, wherein the artery or vein is obstructed by intimalhyperplasia.

In a 401^(st) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 400^(th) embodiments, wherein the pharmaceuticalcomposition or liquid formulation is administered to an obstructedcoronary or peripheral artery.

In a 402^(nd) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 395^(th) embodiments, wherein the artery or vein issusceptible to obstruction by intimal hyperplasia.

In a 403^(rd) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 400^(th) and 402^(nd) embodiments, wherein thecomposition is administered to the wall of a vein.

In a 404^(th) embodiment, the disclosure provides a method of the403^(rd) embodiment, wherein the vein is connected to an arteriovenoushemodialysis graft or arteriovenous fistula.

In a 405^(th) embodiment, the disclosure provides a method of the404^(th) embodiment, wherein the vein is for use in hemodialysis.

In a 406^(th) embodiment, the disclosure provides a method of the405^(th) embodiment, further comprising directly connecting the vein toan artery or connecting the vein to an artery via a graft.

In a 407^(th) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 394^(th) embodiments, wherein the composition isadministered to the adventitial surface of a surgically exposed arteryor vein.

In a 408^(th) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 407^(th) embodiments, wherein the mature type I elastaseprotein in said pharmaceutical composition, liquid formulation orformulation, respectively, is a human type I mature elastase protein.

In a 409^(th) embodiment, the disclosure provides a method of the408^(th) embodiment, wherein the mature human type I elastase proteinconsists essentially of SEQ ID NO:5, SEQ ID NO:84, or SEQ ID NO:87.

In a 410^(th) embodiment, the disclosure provides a method of the408^(th) embodiment, wherein the mature human type I elastase proteinconsists essentially of SEQ ID NO:1 or SEQ ID NO:4.

In a 411^(th) embodiment, the disclosure provides a method of any one ofthe 387^(th) to 407^(th) embodiments, wherein the mature type I elastaseprotein in said pharmaceutical composition, liquid formulation orformulation, respectively, is a porcine type I mature elastase protein.

In a 412^(th) embodiment, the disclosure provides a method of the411^(th) embodiment, wherein the mature porcine type I elastase proteinconsists essentially of SEQ ID NO:39.

In a 413^(th) embodiment, the disclosure provides a unit dosagecomprising 0.0033 mg to 200 mg of (a) mature human type I elastase ofthe 273^(rd) embodiment or the 274^(th) embodiment or (b) a formulationof mature human type I elastase produced by or obtainable by the methodof the 259^(th) embodiment.

In a 414^(th) embodiment, the disclosure provides a unit dosage of the413^(th) embodiment, wherein the mature human type I elastase proteinconsists essentially of SEQ ID NO:5, SEQ ID NO:84, or SEQ ID NO:87.

In a 415^(th) embodiment, the disclosure provides a unit dosagecomprising 0.0033 mg to 200 mg of (a) the mature porcine type I elastaseof the 275^(th) embodiment or the 276^(th) embodiment or (b) aformulation of mature porcine type I elastase produced by or obtainableby the method of the 260^(th) embodiment.

In a 416^(th) embodiment, the disclosure provides a unit dosage of the415^(th) embodiment, wherein the mature porcine type I elastase proteinconsists essentially of SEQ ID NO:39.

In a 417^(th) embodiment, the disclosure provides a unit dosage of anyone of the 413^(th) to 416^(th) embodiments which comprises 0.5 mg to 50mg of said mature type I elastase.

In a 418^(th) embodiment, the disclosure provides a unit dosage of the417^(th) embodiment which comprises 1 mg to 20 mg of said mature type Ielastase.

In a 419^(th) embodiment, the disclosure provides a unit dosage of the418^(th) embodiment which comprises 5 mg to 10 mg of said mature type Ielastase.

In a 420^(th) embodiment, the disclosure provides a unit dosage of anyone of the 413^(th) to 419^(th) embodiments which is in a container,pack, dispenser, or catheter.

In a 421^(st) embodiment, the disclosure provides a kit comprising anelastase protein according to any one of the 1^(st) to 39^(th) and68^(th) to 69^(th) embodiments or obtained or obtainable by the methodof any one of the 89^(th) to 224^(th), 261^(st) to 276^(th), and347^(th) to 373^(rd) embodiments, a nucleic acid according to any one ofthe 40^(th) to 67^(th) embodiments, a vector according to any one of the70^(th) to 72^(nd) embodiments, a cell according to any one of the73^(rd) to 87^(th) embodiments, a cell culture supernatant according tothe 88^(th) embodiment, an elastase formulation according to any one ofthe 314^(th) to 346^(th) embodiments or obtained or obtainable by themethod of any one of the 261^(st) to 276^(th) embodiments, apharmaceutical composition according to the method of any one of the277^(th) to 313^(th) and 386^(th) embodiments, or obtained or obtainableby the method of any one of the 374^(th) to 385^(th) embodiments, or aunit dosage according to any one of the 413^(th) to 420^(th)embodiments.

In a 422^(nd) embodiment, the disclosure provides a kit of the 421^(st)embodiment which is a therapeutic kit.

In a 423^(rd) embodiment, the disclosure provides a kit of the 422^(nd)embodiment which comprises a container, pack, dispenser, or catheter.

In a 424^(th) embodiment, the disclosure provides a kit of the 423^(rd)embodiment which is a manufacturing kit.

The invention is further exemplified by the following SpecificEmbodiments that pertain to proelastase proteins of SEQ ID NO:64 and SEQID NO:69:

In a 1^(st) Specific Embodiment, the disclosure provides a proteincomprising the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69.

In a 2^(nd) Specific Embodiment, the disclosure provides a protein ofthe 1^(st) Specific Embodiment which is isolated.

In a 3^(rd) Specific Embodiment, the disclosure provides a nucleic acidmolecule comprising a nucleotide sequence encoding a protein of the1^(st) Specific Embodiment.

In a 4^(th) Specific Embodiment, the disclosure provides a nucleic acidmolecule of the 3^(rd) Specific Embodiment wherein the protein comprisesa signal sequence operably linked to said amino acid sequence of SEQ IDNO:64 or SEQ ID NO:69.

In a 5^(th) Specific Embodiment, the disclosure provides a nucleic acidmolecule of the 4^(th) Specific Embodiment, wherein the signal sequenceis operable in Pichia pastoris.

In a 6^(th) Specific Embodiment, the disclosure provides a nucleic acidmolecule of the 5^(th) Specific Embodiment, wherein the signal sequenceis a yeast α-factor signal peptide.

In a 7^(th) Specific Embodiment, the disclosure provides a vectorcomprising the nucleic acid molecule of the 4^(th) Specific Embodiment.

In an 8^(th) Specific Embodiment, the disclosure provides a vector ofthe 7^(th) Specific Embodiment in which the nucleotide sequence ismultimerized.

In a 9^(th) Specific Embodiment, the disclosure provides a host cellcomprising the vector of the 7^(th) Specific Embodiment.

In a 10^(th) Specific Embodiment, the disclosure provides a host cell ofthe 9^(th) Specific Embodiment in which at least one copy of said vectoris integrated into the host cell genome.

In a 11^(th) Specific Embodiment, the disclosure provides a host cell ofthe 10^(th) Specific Embodiment in which two to five copies of saidvector are integrated into the host cell genome.

In a 12^(th) Specific Embodiment, the disclosure provides a host cell ofthe 9^(th) Specific Embodiment in which the nucleotide sequence ismultimerized.

In a 13^(th) Specific Embodiment, the disclosure provides a host cell ofthe 12^(th) Specific Embodiment in which the vector comprises two tofive copies of said nucleotide sequence.

In a 14^(th) Specific Embodiment, the disclosure provides a cellgenetically engineered to express the nucleic acid molecule of the3^(rd). Specific Embodiment.

In a 15^(th) Specific Embodiment, the disclosure provides a cell of the14^(th) Specific Embodiment, which is a Pichia pastoris cell.

In a 16^(th) Specific Embodiment, the disclosure provides a cell of the15^(th) Specific Embodiment, wherein the nucleotide sequence is operablylinked to a methanol-inducible promoter.

In a 17^(th) Specific Embodiment, the disclosure provides a cell culturesupernatant comprising the protein of the 1^(st) Specific Embodiment.

In a 18^(th) Specific Embodiment, the disclosure provides a method ofproducing an elastase protein, comprising culturing the cell of the15^(th) Specific Embodiment under conditions in which the protein of SEQID NO:64 or SEQ ID NO:69 is expressed.

In a 19^(th) Specific Embodiment, the disclosure provides a method ofthe 18^(th) Specific Embodiment, wherein said conditions include one,two, three or all four of: (i) a period of growth or induction at a pHof 2 to 6; (ii) a period of growth or induction at a temperature of 22°C. to 28° C.; (iii) culturing in complex medium; or (iv) culturing inthe presence of a citrate, succinate or acetate compound.

In a 20^(th) Specific Embodiment, the disclosure provides a method ofthe 18^(th) Specific Embodiment which further comprises recovering theprotein.

In a 21^(st) Specific Embodiment, the disclosure provides a method ofthe 18^(th) Specific Embodiment, which further comprises exposing saidprotein of SEQ ID NO:64 or SEQ ID NO:69 to activation conditions toproduce a mature elastase protein.

In a 22^(nd) Specific Embodiment, the disclosure provides a method ofthe 21^(st) Specific Embodiment, wherein said protein is purified priorto said exposure to activating conditions.

In a 23^(rd) Specific Embodiment, the disclosure provides a method ofthe 22^(nd) Specific Embodiment, wherein said protein is purified in thepresence of a citrate, succinate or acetate compound.

In a 24^(th) Specific Embodiment, the disclosure provides a method ofthe 21^(st) Specific Embodiment, further comprising purifying the matureelastase protein.

In a 25^(th) Specific Embodiment, the disclosure provides a method ofthe 24^(th) Specific Embodiment, further comprising lyophilizing thepurified mature elastase protein.

In a 26^(th) Specific Embodiment, the disclosure provides a method ofmaking a mature elastase protein, comprising subjecting a cell culturesupernatant according to the 17^(th) Specific Embodiment toautoactivation conditions, thereby producing a mature elastase protein.

In a 27^(th) Specific Embodiment, the disclosure provides a method ofthe 26^(th) Specific Embodiment, further comprising purifying the matureelastase protein.

In a 28^(th) Specific Embodiment, the disclosure provides a method ofthe 27^(th) Specific Embodiment, further comprising lyophilizing thepurified mature elastase protein.

In a 29^(th) Specific Embodiment, the disclosure provides a method ofmaking a pharmaceutical composition comprising a mature elastaseprotein, reconstituting a lyophilisate comprising the lyophilizedproelastase protein produced by the method of the 25^(th) SpecificEmbodiment-or the 28^(th) Specific Embodiment.

In a 30^(th) Specific Embodiment, the disclosure provides a method ofthe 29^(th) Specific Embodiment, wherein the lyophilisate (a) comprisesone or more buffer ingredients or (b) does not comprise bufferingredients.

In a 31^(st) Specific Embodiment, the disclosure provides a method ofthe 30^(th) Specific Embodiment, wherein the lyophilisate isreconstituted with water or buffer.

In a 32^(nd) Specific Embodiment, the disclosure provides a method ofthe 31^(st) Specific Embodiment, wherein upon reconstitution a solutionof mature elastase protein in full strength buffer, greater than fullstrength buffer, or less than full strength buffer is produced.

In a 33^(rd) Specific Embodiment, the disclosure provides a method ofthe 32^(nd) Specific Embodiment, wherein the buffer is phosphatebuffered saline.

In a 34^(th) Specific Embodiment, the disclosure provides a method ofthe 29^(th) Specific Embodiment, wherein the mature elastase protein isreconstituted to a concentration of 0.001 mg/ml to 50 mg/ml.

In a 35^(th) Specific Embodiment, the disclosure provides a method ofthe 29^(th) Specific Embodiment, wherein the mature elastase protein hasa specific activity of 1 to 40 U/mg protein.

In a 36^(th) Specific Embodiment, the disclosure provides apharmaceutical composition produced by the method of the 29^(th)Specific Embodiment.

In a 37^(th) Specific Embodiment, the disclosure provides apharmaceutical composition of the 36^(th) Specific Embodiment which ischaracterized by at least one, at least two, at least three, at leastfour, at least five, at least six or at least seven of the followingproperties:

-   -   (a) the composition is free of trypsin;    -   (b) the composition is substantially free of trypsin;    -   (c) the composition is free of any protein consisting of SEQ ID        NOS:70 and 71;    -   (d) the composition is substantially free of any protein        consisting of SEQ ID NOS:2 and 3;    -   (e) the composition is free of bacterial proteins;    -   (f) the composition is substantially free of bacterial proteins;    -   (g) the composition is free of mammalian proteins other than        said mature elastase protein;    -   (h) the composition is substantially free of mammalian proteins        other than said mature elastase protein;    -   (j) the composition is free or substantially free of one, two,        three or all four proteins consisting of SEQ ID NO:85, 86, 94        and 95;    -   (j) the composition is free or substantially free of one, two,        or all three proteins consisting of SEQ ID NO:106, 107 and 108;    -   (k) the composition contains pharmaceutically acceptable levels        of endotoxins;    -   (l) the mature elastase protein in the composition is        characterized by a specific activity of 1 to 40 U/mg of protein;    -   (m) the trypsin activity in said composition corresponds to less        than 4 ng per 1 mg of mature elastase protein;    -   (n) the composition comprises polysorbate-80;    -   (o) the composition comprises dextran;    -   (p) the composition comprises sodium ions, potassium ions,        phosphate ions, chloride ions and polysorbate-80;    -   (q) the composition comprises sodium ions, potassium ions,        phosphate ions, chloride ions and dextran;    -   (r) the composition comprises sodium ions, potassium ions,        phosphate ions, chloride ions, polysorbate-80, and dextran;    -   (s) the mature elastase protein in said composition maintains        60% to 100% of its specific activity after at least a week of        storage at 4° C., after at least a month of storage at 4° C.,        after at least two months of storage at 4° C., after at least        three months of storage at 4° C., or after at least month six        months of storage at 4° C.; and    -   (t) the composition comprises a unit dosage of 0.0033 mg to 200        mg of said mature elastase protein.

In a 38^(th) Specific Embodiment, the disclosure provides apharmaceutical composition of the 37^(th) Specific Embodiment, whereinthe pharmaceutical composition is characterized by at least threecharacteristics, at least four characteristics or five characteristicsindependently selected from the following groups (i) through (v):

-   -   (i) (a), (b) or (m)    -   (ii) (e) or (f)    -   (iii) (g) or (h)    -   (iv) (k)    -   (v) (l)

In a 39^(th) Specific Embodiment, the disclosure provides apharmaceutical composition of the 38^(th) Specific Embodiment, whereintwo of said at least three or at least said four characteristics areselected from groups (i) and (iv) or (v).

In a 40^(th) Specific Embodiment, the disclosure provides apharmaceutical composition of the 38^(th) Specific Embodiment, whereinthree of at least said four characteristics are selected from groups(i), (iv) and (v).

In a 41^(st) Specific Embodiment, the disclosure provides a method ofremoving one or more incorrectly processed mature elastase proteins froma mixture of correctly and incorrectly processed mature elastaseproteins, said method comprising:

-   -   (a) subjecting a composition comprising a mixture of correctly        and incorrectly processed mature elastase proteins to a pH at        which the correctly processed mature enzyme is active;    -   (b) maintaining such pH until such time that one or more        incorrectly processed mature elastase proteins are degraded,

thereby removing said one or more incorrectly processed mature elastaseproteins from a mixture of correctly and incorrectly processed matureelastase proteins.

In a 42^(nd) Specific Embodiment, the disclosure provides a method ofthe 41^(st) Specific Embodiment, wherein said one or more incorrectlyprocessed mature elastase proteins contain at least one additional orfewer amino acid at the N-terminus relative to correctly processedmature elastase proteins.

In a 43^(rd) Specific Embodiment, the disclosure provides a method ofthe 41^(st) Specific Embodiment wherein said pH is between 5 and 12.

In a 44^(th) Specific Embodiment, the disclosure provides a method ofany one of the 41^(st) Specific Embodiments, wherein said one or moreincorrectly processed mature elastase proteins are degraded by 50% to100%.

In a 45^(th) Specific Embodiment, the disclosure provides a method fortherapeutically increasing the diameter of an artery or vein in a humansubject in need thereof, the method comprising: locally administering tothe wall of the artery or vein in the human subject the pharmaceuticalcomposition of the 36^(th) Specific Embodiment in a dose sufficient toincrease the diameter of the artery or vein.

In a 46^(th) Specific Embodiment, the disclosure provides a method ofthe 45^(th) Specific Embodiment, wherein the diameter of the vessel, thelumenal diameter of the vessel, or both, are increased.

In a 47^(th) Specific Embodiment, the disclosure provides a method forpreventing or treating vasospasm of an artery or vein in a human subjectin need thereof, the method comprising: locally administering to thewall of the artery or vein in the human subject the pharmaceuticalcomposition of the 36^(th) Specific Embodiment in a dose sufficient toprevent or treat vasospasm of the artery or vein.

In a 48^(th) Specific Embodiment, the disclosure provides a method fortreating an obstructed artery or vein in a human subject in need of suchtreatment, the method comprising: locally administering to the wall ofthe artery or vein in the human subject the pharmaceutical compositionof the 36^(th) Specific Embodiments, wherein said administration resultsin proteolysis of elastin in the wall of the artery or vein leading toenlargement of the diameter of the artery or vein.

In a 49^(th) Specific Embodiment, the disclosure provides a method fortreating an artery or vein connected to an arteriovenous hemodialysisgraft or arteriovenous fistula in a human subject in need of suchtreatment, the method comprising: locally administering to the wall ofthe artery or vein in the human subject the pharmaceutical compositionof the 36^(th) Specific Embodiment, wherein said administration resultsin proteolysis of elastin in the wall of the artery or vein leading toenlargement of the diameter of the artery or vein.

In a 50^(th) Specific Embodiment, the disclosure provides a method fortreating a vein in a human subject for use in hemodialysis, the methodcomprising: locally administering to the wall of the vein in the humansubject the pharmaceutical composition of the 36^(th) SpecificEmbodiment, wherein said administration results in proteolysis ofelastin in the wall of the vein leading to enlargement of the diameterof the vein.

In a 51^(st) Specific Embodiment, the disclosure provides a kitcomprising the pharmaceutical composition of the 36^(th) SpecificEmbodiment.

In a 52^(nd) Specific Embodiment, the disclosure provides a kit of the51^(st) Specific Embodiment wherein the pharmaceutical composition is ina container, pack, dispenser, or catheter.

The claims of U.S. provisional application No. 60/992,319, filed Dec. 4,2007 are incorporated by reference herein in their entireties, and eachembodiment set forth in such claims is incorporated by reference as aspecific embodiment herein.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references, including patent applications, patents, andscientific publications, are cited herein; the disclosure of each suchreference is hereby incorporated herein by reference in its entirety.

1. A proelastase protein comprising (i) optionally, a signal sequence;(ii) an elastase activation peptide sequence comprising an elastaserecognition sequence operably linked to (iii) an amino acid sequence ofa type I mature elastase.
 2. The proelastase protein of claim 1, whereinsaid elastase recognition sequence comprises the amino acid sequence ofSEQ ID NO:119.
 3. The proelastase protein of claim 2, wherein proline isat the P2 position of said elastase recognition sequence.
 4. Theproelastase protein of claim 1, wherein said elastase recognitionsequence comprises the amino acid sequence of SEQ ID NO:124.
 5. Theproelastase protein of claim 4, wherein proline is at the P2 position ofsaid elastase recognition sequence.
 6. The proelastase protein of claim1 which comprises a cleavage domain the propeptide portion of whichcomprises the amino acid sequence of SEQ ID NO:120.
 7. The proelastaseprotein of claim 4, wherein proline is at the P2 position of saidcleavage domain.
 8. The proelastase protein of claim 4, whereinhistidine is at the P5 position of said cleavage domain.
 9. Theproelastase protein of claim 1 which comprises a cleavage domain of SEQID NO:123 or SEQ ID NO:125.
 10. The proelastase protein of claim 1,wherein said activation peptide sequence comprises the amino acidsequence of SEQ ID NO:121.
 11. The proelastase protein of claim 1,wherein the amino acid sequence of said type I mature elastase has atleast 90% sequence identity to the amino acid sequence from position 6to the end of SEQ ID NO:84 or SEQ ID NO:1.
 12. The proelastase proteinof claim 1 which comprises a signal sequence.
 13. (canceled)
 14. Anucleic acid encoding a proelastase protein of claim
 1. 15. A vectorcomprising the nucleic acid molecule of claim
 14. 16. A host cellgenetically engineered to express the nucleic acid molecule of claim 14.17. A solution comprising the proelastase protein of any one of claim 1.18-20. (canceled)
 21. A cell culture supernatant comprising theproelastase protein of claim
 1. 22. A method of producing a proelastaseprotein, comprising culturing the host cell of claim 16 under conditionsin which the proelastase protein is produced.
 23. (canceled)
 24. Amethod of producing a mature elastase protein, comprising subjecting thesolution of claim 17 to conditions that produce mature elastase protein.25-28. (canceled)
 29. A method of producing a mature elastase protein,comprising: (a) culturing the host cell of claim 16 under conditions inwhich the proelastase protein is produced; (b) recovering theproelastase protein, and (c) subjecting a solution comprising saidproelastase protein to conditions that produce a mature elastaseprotein, thereby producing a mature elastase protein. 30-32. (canceled)